Peptidomimetic macrocycles

ABSTRACT

Biologically active crosslinked polypeptides with improved properties relative to their corresponding precursor polypeptides, having good cell penetration properties and reduced binding to human proteins, and methods of identifying and making such improved polypeptides are described.

CROSS REFERENCE

This application claims the benefit of U.S. Provisional Application No.61/099,063, entitled “Organic Compounds” filed Sep. 22, 2008, which isincorporated herein in its entirety by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Feb. 21, 2012, isnamed 35224202.txt and is 63,708 bytes in size.

BACKGROUND OF THE INVENTION

Recombinant or synthetically produced peptides have importantapplications as pharmaceuticals. Peptides, however, often suffer frompoor metabolic stability, poor cell penetrability, and promiscuousbinding due to conformational flexibility. One approach to stabilizingthese peptides is to use intramolecular crosslinkers to maintain thepeptide in the desired configuration, for example using disulfide bonds,amide bonds, or carbon-carbon bonds to link amino acid side chains. See,e.g., Jackson et al. (1991), J. Am. Chem. Soc. 113:9391-9392; Phelan etal. (1997), J. Am. Chem. Soc. 119:455-460; Taylor (2002), Biopolymers66: 49-75; Brunel et al. (2005), Chem. Commun. (20):2552-2554; Hiroshigeet al. (1995), J. Am. Chem. Soc. 117: 11590-11591; Blackwell et al.(1998), Angew. Chem. Int. Ed. 37:3281-3284; Schafmeister et al. (2000),J. Am. Chem. Soc. 122:5891-5892; Walensky et al. (2004), Science305:1466-1470; Bernal et al. (2007), J. Am. Chem Soc. 129:2456-2457;U.S. Pat. No. 7,192,713 B1 (Verdine et al) (describing cross-linkedstabilized-helical peptides comprising natural and non-natural aminoacids, wherein the peptide comprises at least two reactive moietiescapable of undergoing a C—C bond-forming reaction); and U.S. Pat. No.5,811,515 (Grubbs et al) (describing the synthesis ofconformationally-restricted/cyclic-stabilized peptides andpeptidomimetics from precursors containing two or more unsaturated C—Cbonds); the contents of which patents and publications are incorporatedherein by reference. Such polypeptides which are conformationallystabilized by means of intramolecular cross-linkers are sometimesreferred to as “stapled” polypeptides.

A major advantage of these crosslinked polypeptides is that they have anenhanced ability to penetrate cell membranes relative to theirnon-stapled counterparts. This cellular uptake is believed to bemediated by an active transport mechanism utilizing endocytosis.

Some of the physical characteristics which facilitate the entry of thepeptides into the cells also tend to increase the affinity ofcrosslinked peptides to serum proteins, such as albumin. Consequently,many highly promising leads exhibit a marked “serum shift”, havinggreatly diminished activity in vivo or in assays having serum basedmedia, compared to activity in assays using serum-free media, renderingthe peptides less than optimal for therapeutic or diagnosticapplications. Crosslinked polypeptides having low levels of serumbinding, however, tend to have poor cell penetration, as well as poorpharmacokinetics, e.g., rapid renal or first pass clearance. Thisinvention addresses this and other problems.

SUMMARY OF THE INVENTION

The invention discloses methods for the identification and optimizationof crosslinked polypeptides that possess reduced affinity to serumproteins to permit good activity in the presence of serum, while stillretaining sufficient affinity to the cell membranes to be readilytransported into the cell, retaining sufficient affinity to serumproteins to have acceptable pharmacokinetics, and retaining highaffinity binding to target receptor(s) within the cell. The inventorshave discovered that there is an optimal range of serum protein bindingfor crosslinked polypeptides for achieving these objectives. Theinvention further provides optimal compounds with superior cellpenetration and biological activities in the presence of serum, andstructure-activity relationships to permit optimization of crosslinkedpolypeptides having improved therapeutic efficacies or diagnosticactivities.

In one embodiment, the present invention provides a method ofidentifying cross-linked polypeptides with improved efficacies in humanwhole blood, comprising the steps of synthesizing analogs of the parentcross-linked polypeptide and performing cellular assays in the absenceof human serum proteins and also in the presence of one or moreconcentrations of human serum, so as to determine the apparent affinityof each cross-linked polypeptide for human serum proteins.

In another embodiment, the present invention provides a method ofpreparing a polypeptide with optimized cellular efficacy in human wholeblood, the method comprising: a) providing a parent polypeptidecomprising a cross-linker connecting a first amino acid and a secondamino acid of said polypeptide, and wherein the parent polypeptidepenetrates cell membranes by an energy-dependent process and binds to anintracellular target; b) identifying one or more dipeptide motifs insaid parent polypeptide consisting of an acidic side chain adjacent to alarge hydrophobic side chain, wherein the acidic side chain is notessential to binding the target; c) replacing the acidic side chain insaid motif with a neutral side chain to prepare a modified parentpolypeptide; d) measuring the in vitro efficacies of the modified parentpolypeptide polypeptides in a whole cell assay wherein activity ismediated by binding to the intracellular target, in the presence andabsence of human serum; e) calculating the apparent affinity (K_(d)*) ofthe modified polypeptide to human serum proteins and its EC₅₀; and f)selecting the modified parent polypeptide as an optimized polypeptide ifsaid modified parent polypeptide has a higher K_(d)* and an equal orlower EC₅₀ than the parent polypeptide. In some embodiments, K_(d)* isdefined by the equation

${EC}_{50}^{\prime} = {{EC}_{50} + {P\left( \frac{n}{1 + \frac{K_{d}^{*}}{{EC}_{50}}} \right)}}$where n is 1, EC₅₀ is an in vitro efficacy measured in a whole cellassay in the absence of any human serum, and EC′₅₀ is an in vitroefficacy measured in a whole cell assay in N % human serum wherein Pequals (N/100)×(700) micromolar.

In some embodiments of the method, both the acidic and large hydrophobicside chains in said dipeptide motif are not essential to binding thetarget and are replaced with neutral and less hydrophobic side chains,respectively.

For example, the invention provides a method of screening a polypeptidecomprising a cross-linker connecting a first amino acid and a secondamino acid of said polypeptide, wherein the polypeptide penetrates cellmembranes by an energy-dependent process and binds to an intracellulartarget, the method comprising measuring the in vitro efficacy of thepolypeptide in a whole cell assay in the presence and absence of humanserum; calculating the apparent affinity (K_(d)*) of the polypeptide tohuman serum proteins, wherein K_(d)* is defined by the equation

${EC}_{50}^{\prime} = {{EC}_{50} + {P\left( \frac{n}{1 + \frac{K_{d}^{*}}{{EC}_{50}}} \right)}}$where n is 1, EC₅₀ is an in vitro efficacy measured in a whole cellassay in the absence of any human serum, EC′₅₀ is an in vitro efficacymeasured in a whole cell assay in N % human serum wherein P equals(N/100)×(700) micromolar; and selecting compounds having a K_(d)* offrom 1 to 700 micromolar, e.g., 1-70 micromolar, for example 10-70micromolar. For example, the selected compound may possess an estimatedfree fraction in human blood of 0.1-50%, e.g. 0.5-10% wherein theestimated free fraction is defined by the equation

${FreeFraction} = \frac{K_{d}^{*}}{K_{d}^{*} + \lbrack{HSA}\rbrack_{total}}$and [HSA]_(total) is 700 micromolar.

In some embodiments of the method, the biological activity (EC50) ismeasured as the percentage of the number of cells killed in an in vitroassay in which cultured cells are exposed to an effective concentrationof said polypeptide.

In some embodiments, the polypeptide is selected such that the apparentserum binding affinity (Kd*) of the crosslinked polypeptide is 1, 3, 10,70 micromolar or greater. In other embodiments, the Kd* of thecrosslinked polypeptide is 1 to 10, 70, or 700 micromolar. In otherembodiments, the crosslinked polypeptides is selected such that itpossesses an estimated free fraction in human blood of between 0.1 and50%, or between 0.15 and 10%.

The invention further provides polypeptides selected using the methodsof the invention, or otherwise meeting the criteria of the invention.For example, in some embodiments, the improved cross-linked polypeptidepossesses an apparent affinity to human serum proteins of 1 micromolaror weaker. In another embodiment, the improved cross-linked polypeptidepossesses an apparent affinity to human serum proteins of 3 micromolaror weaker. In another embodiment, the improved cross-linked polypeptidepossesses an apparent affinity to human serum proteins of 10 micromolaror weaker. In another embodiment, the improved cross-linked polypeptidepossesses an apparent affinity to human serum proteins of 70 micromolaror weaker. In another embodiment, the improved cross-linked polypeptidepossesses an apparent affinity to human serum proteins of between 1-70micromolar. In another embodiment, the improved cross-linked polypeptidepossesses an apparent affinity to human serum proteins of between 1-700micromolar. In some embodiments, the improved cross-linked polypeptidepossesses an estimated free fraction in whole blood of between 0.1-50%.In another embodiment, the improved cross-linked polypeptide possessesan estimated free fraction in whole blood of between 0.5-10%.

In some embodiments of the method, said polypeptide contains onecrosslink. In other embodiments of the method, said polypeptide containstwo cross-links.

In some embodiments of the method, one crosslink connects two α-carbonatoms. In other embodiments of the method, one α-carbon atom to whichone crosslink is attached is substituted with a substituent of formulaR—. In another embodiment of the method, two α-carbon atoms to which onecrosslink is attached are substituted with independent substituents offormula R—.

In one embodiment of the methods of the invention, R— is alkyl. Forexample, R— is methyl. Alternatively, R— and any portion of onecrosslink taken together can form a cyclic structure. In anotherembodiment of the method, one crosslink is formed of consecutivecarbon-carbon bonds. For example, one crosslink may comprise at least 8,9, 10, 11, or 12 consecutive bonds. In other embodiments, one crosslinkmay comprise at least 7, 8, 9, 10, or 11 carbon atoms.

In some embodiments of the method, the crosslinked polypeptidepenetrates cell membranes by an energy-dependent process and binds to anintracellular target.

In another embodiment, the improved crosslinked polypeptide comprises anα-helical domain of a BCL-2 family member. For example, the crosslinkedpolypeptide comprises a BH3 domain. In other embodiments, thecrosslinked polypeptide have a sequence identity of at least 60%, 70%,80%, 85%, 90% or 95% to any of the sequences in Tables 1, 2, 3 and 4,e.g., as measured in a BLAST algorithm.

The invention further provides methods of using the improved crosslinkedpolypeptides of the invention in prophylactic and therapeutic methods oftreating a subject at risk of (or susceptible to) a disorder or having adisorder associated with aberrant (e.g., insufficient or excessive)BCL-2 family member expression or activity (e.g., extrinsic or intrinsicapoptotic pathway abnormalities); and for treating or preventinghyperproliferative disease by interfering with the interaction orbinding between p53 and MDM2 in hyperproliferative cells, e.g. tumorcells.

Further aspects of the invention will be apparent from the detaileddescription, the examples, the drawings and the claims below.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 shows peptidomimetic macrocycle (compound 1) dose response curvesin the presence of varying concentrations of human serum.

FIG. 2 shows a plot of cellular EC₅₀ vs human serum concentrations forpeptidomimetic macrocycle analogs with improved properties.

FIG. 3 shows a plot of cellular EC₅₀ vs human serum concentrations forpeptidomimetic macrocycle analogs with improved properties.

FIG. 4 shows helical wheel representations of improved peptidomimeticmacrocycle analogs (SEQ ID NOS 116-118 and 122, respectively, in orderof appearance).

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the term “macrocycle” refers to a molecule having achemical structure including a ring or cycle formed by at least 9covalently bonded atoms.

As used herein, the term “stapled polypeptide” or “crosslinkedpolypeptide” refers to a compound comprising a plurality of amino acidresidues joined by a plurality of peptide bonds and at least onemacrocycle-forming linker which forms a macrocycle between a firstnaturally-occurring or non-naturally-occurring amino acid residue (oranalog) and a second naturally-occurring or non-naturally-occurringamino acid residue (or analog) within the same molecule. Crosslinkedpolypeptide include embodiments where the macrocycle-forming linkerconnects the α carbon of the first amino acid residue (or analog) to theα carbon of the second amino acid residue (or analog). The crosslinkedpolypeptides optionally include one or more non-peptide bonds betweenone or more amino acid residues and/or amino acid analog residues, andoptionally include one or more non-naturally-occurring amino acidresidues or amino acid analog residues in addition to any which form themacrocycle.

As used herein, the term “stability” refers to the maintenance of adefined secondary structure in solution by a crosslinked polypeptide ofthe invention as measured by circular dichroism, NMR or anotherbiophysical measure, or resistance to proteolytic degradation in vitroor in vivo. Non-limiting examples of secondary structures contemplatedin this invention are α-helices, β-turns, and β-pleated sheets.

As used herein, the term “helical stability” refers to the maintenanceof α helical structure by a crosslinked polypeptide of the invention asmeasured by circular dichroism or NMR. For example, in some embodiments,the crosslinked polypeptides of the invention exhibit at least a 1.25,1.5, 1.75 or 2-fold increase in α-helicity as determined by circulardichroism compared to a corresponding macrocycle lacking the R—substituent.

The term “α-amino acid” or simply “amino acid” refers to a moleculecontaining both an amino group and a carboxyl group bound to a carbonwhich is designated the α-carbon. Suitable amino acids include, withoutlimitation, both the D- and L-isomers of the naturally-occurring aminoacids, as well as non-naturally occurring amino acids prepared byorganic synthesis or other metabolic routes. Unless the contextspecifically indicates otherwise, the term amino acid, as used herein,is intended to include amino acid analogs.

The term “naturally occurring amino acid” refers to any one of thetwenty amino acids commonly found in peptides synthesized in nature, andknown by the one letter abbreviations A, R, N, C, D, Q, E, G, H, I, L,K, M, F, P, S, T, W, Y and V.

The term “amino acid analog” or “non-natural amino acid” refers to amolecule which is structurally similar to an amino acid and which can besubstituted for an amino acid in the formation of a crosslinkedpolypeptide. Amino acid analogs include, without limitation, compoundswhich are structurally identical to an amino acid, as defined herein,except for the inclusion of one or more additional methylene groupsbetween the amino and carboxyl group (e.g., α-amino β-carboxy acids), orfor the substitution of the amino or carboxy group by a similarlyreactive group (e.g., substitution of the primary amine with a secondaryor tertiary amine, or substitution or the carboxy group with an ester).

A “non-essential” amino acid residue is a residue that can be alteredfrom the wild-type sequence of a polypeptide (e.g., a BH3 domain or thep53 MDM2 binding domain) without abolishing or substantially alteringits essential biological or biochemical activity (e.g., receptor bindingor activation). An “essential” amino acid residue is a residue that,when altered from the wild-type sequence of the polypeptide, results inabolishing or substantially abolishing the polypeptide's essentialbiological or biochemical activity.

A “conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art. These families include amino acids with basicside chains (e.g., K, R, H), acidic side chains (e.g., D, E), unchargedpolar side chains (e.g., G, N, Q, S, T, Y, C), nonpolar side chains(e.g., A, V, L, I, P, F, M, W), beta-branched side chains (e.g., T, V,I) and aromatic side chains (e.g., Y, F, W, H). Thus, a predictednonessential amino acid residue in a BH3 polypeptide, for example, ispreferably replaced with another amino acid residue from the same sidechain family. Other examples of acceptable substitutions aresubstitutions based on isosteric considerations (e.g. norleucine formethionine) or other properties (e.g. 2-thienylalanine forphenylalanine).

The term “member” as used herein in conjunction with macrocycles ormacrocycle-forming linkers refers to the atoms that form or can form themacrocycle, and excludes substituent or side chain atoms. By analogy,cyclodecane, 1,2-difluoro-decane and 1,3-dimethyl cyclodecane are allconsidered ten-membered macrocycles as the hydrogen or fluorosubstituents or methyl side chains do not participate in forming themacrocycle.

The symbol

when used as part of a molecular structure refers to a single bond or atrans or cis double bond.

The term “amino acid side chain” refers to a moiety attached to theα-carbon in an amino acid. For example, the amino acid side chain foralanine is methyl, the amino acid side chain for phenylalanine isphenylmethyl, the amino acid side chain for cysteine is thiomethyl, theamino acid side chain for aspartate is carboxymethyl, the amino acidside chain for tyrosine is 4-hydroxyphenylmethyl, etc. Othernon-naturally occurring amino acid side chains are also included, forexample, those that occur in nature (e.g., an amino acid metabolite) orthose that are made synthetically (e.g., an α,α di-substituted aminoacid).

The term “α,α di-substituted amino” acid refers to a molecule or moietycontaining both an amino group and a carboxyl group bound to a carbon(the α-carbon) that is attached to two natural or non-natural amino acidside chains.

The term “polypeptide” encompasses two or more naturally ornon-naturally-occurring amino acids joined by a covalent bond (e.g., anamide bond). Polypeptides as described herein include full lengthproteins (e.g., fully processed proteins) as well as shorter amino acidsequences (e.g., fragments of naturally-occurring proteins or syntheticpolypeptide fragments).

The term “macrocyclization reagent” or “macrocycle-forming reagent” asused herein refers to any reagent which may be used to prepare acrosslinked polypeptide of the invention by mediating the reactionbetween two reactive groups. Reactive groups may be, for example, anazide and alkyne, in which case macrocyclization reagents include,without limitation, Cu reagents such as reagents which provide areactive Cu(I) species, such as CuBr, CuI or CuOTf, as well as Cu(II)salts such as Cu(CO₂CH₃)₂, CuSO₄, and CuCl₂ that can be converted insitu to an active Cu(I) reagent by the addition of a reducing agent suchas ascorbic acid or sodium ascorbate. Macrocyclization reagents mayadditionally include, for example, Ru reagents known in the art such asCp*RuCl(PPh₃)₂, [Cp*RuCl]₄ or other Ru reagents which may provide areactive Ru(II) species. In other cases, the reactive groups areterminal olefins. In such embodiments, the macrocyclization reagents ormacrocycle-forming reagents are metathesis catalysts including, but notlimited to, stabilized, late transition metal carbene complex catalystssuch as Group VIII transition metal carbene catalysts. For example, suchcatalysts are Ru and Os metal centers having a +2 oxidation state, anelectron count of 16 and pentacoordinated. Additional catalysts aredisclosed in Grubbs et al., “Ring Closing Metathesis and RelatedProcesses in Organic Synthesis” Acc. Chem. Res. 1995, 28, 446-452, andU.S. Pat. No. 5,811,515. In yet other cases, the reactive groups arethiol groups. In such embodiments, the macrocyclization reagent is, forexample, a linker functionalized with two thiol-reactive groups such ashalogen groups.

The term “halo” or “halogen” refers to fluorine, chlorine, bromine oriodine or a radical thereof.

The term “alkyl” refers to a hydrocarbon chain that is a straight chainor branched chain, containing the indicated number of carbon atoms. Forexample, C₁-C₁₀ indicates that the group has from 1 to 10 (inclusive)carbon atoms in it. In the absence of any numerical designation, “alkyl”is a chain (straight or branched) having 1 to 20 (inclusive) carbonatoms in it.

The term “alkylene” refers to a divalent alkyl (i.e., —R—).

The term “alkenyl” refers to a hydrocarbon chain that is a straightchain or branched chain having one or more carbon-carbon double bonds.The alkenyl moiety contains the indicated number of carbon atoms. Forexample, C₂-C₁₀ indicates that the group has from 2 to 10 (inclusive)carbon atoms in it. The term “lower alkenyl” refers to a C₂-C₆ alkenylchain. In the absence of any numerical designation, “alkenyl” is a chain(straight or branched) having 2 to 20 (inclusive) carbon atoms in it.

The term “alkynyl” refers to a hydrocarbon chain that is a straightchain or branched chain having one or more carbon-carbon triple bonds.The alkynyl moiety contains the indicated number of carbon atoms. Forexample, C₂-C₁₀ indicates that the group has from 2 to 10 (inclusive)carbon atoms in it. The term “lower alkynyl” refers to a C₂-C₆ alkynylchain. In the absence of any numerical designation, “alkynyl” is a chain(straight or branched) having 2 to 20 (inclusive) carbon atoms in it.

The term “aryl” refers to a 6-carbon monocyclic or 10-carbon bicyclicaromatic ring system wherein 0, 1, 2, 3, or 4 atoms of each ring aresubstituted by a substituent. Examples of aryl groups include phenyl,naphthyl and the like. The term “arylalkyl” or the term “aralkyl” refersto alkyl substituted with an aryl. The term “arylalkoxy” refers to analkoxy substituted with aryl.

“Arylalkyl” refers to an aryl group, as defined above, wherein one ofthe aryl group's hydrogen atoms has been replaced with a C₁-C₅ alkylgroup, as defined above. Representative examples of an arylalkyl groupinclude, but are not limited to, 2-methylphenyl, 3-methylphenyl,4-methylphenyl, 2-ethylphenyl, 3-ethylphenyl, 4-ethylphenyl,2-propylphenyl, 3-propylphenyl, 4-propylphenyl, 2-butylphenyl,3-butylphenyl, 4-butylphenyl, 2-pentylphenyl, 3-pentylphenyl,4-pentylphenyl, 2-isopropylphenyl, 3-isopropylphenyl, 4-isopropylphenyl,2-isobutylphenyl, 3-isobutylphenyl, 4-isobutylphenyl, 2-sec-butylphenyl,3-sec-butylphenyl, 4-sec-butylphenyl, 2-t-butylphenyl, 3-t-butylphenyland 4-t-butylphenyl.

“Arylamido” refers to an aryl group, as defined above, wherein one ofthe aryl group's hydrogen atoms has been replaced with one or more—C(O)NH₂ groups. Representative examples of an arylamido group include2-C(O)NH2-phenyl, 3-C(O)NH₂-phenyl, 4-C(O)NH₂-phenyl, 2-C(O)NH₂-pyridyl,3-C(O)NH₂-pyridyl, and 4-C(O)NH₂-pyridyl,

“Alkylheterocycle” refers to a C₁-C₅ alkyl group, as defined above,wherein one of the C₁-C₅ alkyl group's hydrogen atoms has been replacedwith a heterocycle. Representative examples of an alkylheterocycle groupinclude, but are not limited to, —CH₂CH₂-morpholine, —CH₂CH₂-piperidine,—CH₂CH₂CH₂-morpholine, and —CH₂CH₂CH₂-imidazole.

“Alkylamido” refers to a C₁-C₅ alkyl group, as defined above, whereinone of the C₁-C₅ alkyl group's hydrogen atoms has been replaced with a—C(O)NH₂ group. Representative examples of an alkylamido group include,but are not limited to, —CH₂—C(O)NH₂, —CH₂CH₂—C(O)NH₂,—CH₂CH₂CH₂C(O)NH₂, —CH₂CH₂CH₂CH₂C(O)NH₂, —CH₂CH₂CH₂CH₂CH₂C(O)NH₂,—CH₂CH(C(O)NH₂)CH₃, —CH₂CH(C(O)NH₂)CH₂CH₃, —CH(C(O)NH₂)CH₂CH₃,—C(CH₃)₂CH₂C(O)NH₂, —CH₂—CH₂—NH—C(O)—CH₃, —CH₂—CH₂—NH—C(O)—CH₃—CH3, and—CH₂—CH₂—NH—C(O)—CH═CH₂.

“Alkanol” refers to a C₁-C₅ alkyl group, as defined above, wherein oneof the C₁-C₅ alkyl group's hydrogen atoms has been replaced with ahydroxyl group. Representative examples of an alkanol group include, butare not limited to, —CH₂OH, —CH₂CH₂OH, —CH₂CH₂CH₂OH, —CH₂CH₂CH₂CH₂OH,—CH₂CH₂CH₂CH₂CH₂OH, —CH₂CH(OH)CH₃, —CH₂CH(OH)CH₂CH₃, —CH(OH)CH₃ and—C(CH₃)₂CH₂OH.

“Alkylcarboxy” refers to a C₁-C₅ alkyl group, as defined above, whereinone of the C₁-C₅ alkyl group's hydrogen atoms has been replaced with a—COOH group. Representative examples of an alkylcarboxy group include,but are not limited to, —CH₂COOH, —CH₂CH₂COOH, —CH₂CH₂CH₂COOH,—CH₂CH₂CH₂CH₂COOH, —CH₂CH(COOH)CH₃, —CH₂CH₂CH₂CH₂CH₂COOH,—CH₂CH(COOH)CH₂CH₃, —CH(COOH)CH₂CH₃ and —C(CH₃)₂CH₂COOH.

The term “cycloalkyl” as employed herein includes saturated andpartially unsaturated cyclic hydrocarbon groups having 3 to 12 carbons,preferably 3 to 8 carbons, and more preferably 3 to 6 carbons, whereinthe cycloalkyl group additionally is optionally substituted. Somecycloalkyl groups include, without limitation, cyclopropyl, cyclobutyl,cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, andcyclooctyl.

The term “heteroaryl” refers to an aromatic 5-8 membered monocyclic,8-12 membered bicyclic, or 11-14 membered tricyclic ring system having1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9heteroatoms if tricyclic, said heteroatoms selected from O, N, or S(e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of O, N, or S ifmonocyclic, bicyclic, or tricyclic, respectively), wherein 0, 1, 2, 3,or 4 atoms of each ring are substituted by a substituent. Examples ofheteroaryl groups include pyridyl, furyl or furanyl, imidazolyl,benzimidazolyl, pyrimidinyl, thiophenyl or thienyl, quinolinyl, indolyl,thiazolyl, and the like.

The term “heteroarylalkyl” or the term “heteroaralkyl” refers to analkyl substituted with a heteroaryl. The term “heteroarylalkoxy” refersto an alkoxy substituted with heteroaryl.

The term “heteroarylalkyl” or the term “heteroaralkyl” refers to analkyl substituted with a heteroaryl. The term “heteroarylalkoxy” refersto an alkoxy substituted with heteroaryl.

The term “heterocyclyl” refers to a nonaromatic 5-8 membered monocyclic,8-12 membered bicyclic, or 11-14 membered tricyclic ring system having1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9heteroatoms if tricyclic, said heteroatoms selected from O, N, or S(e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of O, N, or S ifmonocyclic, bicyclic, or tricyclic, respectively), wherein 0, 1, 2 or 3atoms of each ring are substituted by a substituent. Examples ofheterocyclyl groups include piperazinyl, pyrrolidinyl, dioxanyl,morpholinyl, tetrahydrofuranyl, and the like.

The term “substituent” refers to a group replacing a second atom orgroup such as a hydrogen atom on any molecule, compound or moiety.Suitable substituents include, without limitation, halo, hydroxy,mercapto, oxo, nitro, haloalkyl, alkyl, alkaryl, aryl, aralkyl, alkoxy,thioalkoxy, aryloxy, amino, alkoxycarbonyl, amido, carboxy,alkanesulfonyl, alkylcarbonyl, and cyano groups.

In some embodiments, the compounds of this invention contain one or moreasymmetric centers and thus occur as racemates and racemic mixtures,single enantiomers, individual diastereomers and diastereomericmixtures. All such isomeric forms of these compounds are included in thepresent invention unless expressly provided otherwise. In someembodiments, the compounds of this invention are also represented inmultiple tautomeric forms, in such instances, the invention includes alltautomeric forms of the compounds described herein (e.g., if alkylationof a ring system results in alkylation at multiple sites, the inventionincludes all such reaction products). All such isomeric forms of suchcompounds are included in the present invention unless expresslyprovided otherwise. All crystal forms of the compounds described hereinare included in the present invention unless expressly providedotherwise.

As used herein, the terms “increase” and “decrease” mean, respectively,to cause a statistically significantly (i.e., p<0.1) increase ordecrease of at least 5%.

As used herein, the recitation of a numerical range for a variable isintended to convey that the invention may be practiced with the variableequal to any of the values within that range. Thus, for a variable whichis inherently discrete, the variable is equal to any integer valuewithin the numerical range, including the end-points of the range.Similarly, for a variable which is inherently continuous, the variableis equal to any real value within the numerical range, including theend-points of the range. As an example, and without limitation, avariable which is described as having values between 0 and 2 takes thevalues 0, 1 or 2 if the variable is inherently discrete, and takes thevalues 0.0, 0.1, 0.01, 0.001, or any other real values≧0 and ≦2 if thevariable is inherently continuous.

As used herein, unless specifically indicated otherwise, the word “or”is used in the inclusive sense of “and/or” and not the exclusive senseof “either/or.”

The term “on average” represents the mean value derived from performingat least three independent replicates for each data point.

The term “biological activity” encompasses structural and functionalproperties of a macrocycle of the invention. Biological activity is, forexample, structural stability, alpha-helicity, affinity for a target,resistance to proteolytic degradation, cell penetrability, intracellularstability, in vivo stability, or any combination thereof.

The details of one or more particular embodiments of the invention areset forth in the accompanying drawings and the description below. Otherfeatures, objects, and advantages of the invention will be apparent fromthe description and drawings, and from the claims.

Biological Properties of the Crosslinked Polypeptides of the Invention

In one embodiment, the present invention provides a method ofidentifying cross-linked polypeptides with improved efficacies in humanwhole blood, comprising the steps of synthesizing analogs of the parentcross-linked polypeptide and performing cellular assays in the absenceof human serum proteins and also in the presence of two or moreconcentrations of human serum, so as to determine the apparent affinityof each cross-linked polypeptide for human serum proteins and tocalculate an EC50 in whole blood by mathematical extrapolation.

In some embodiments, the polypeptide is selected such that the apparentserum binding affinity (Kd*) of the crosslinked polypeptide is 1, 3, 10,70 micromolar or greater. In other embodiments, the Kd* of thecrosslinked polypeptide is 1 to 10, 70, or 700 micromolar. In otherembodiments, the crosslinked polypeptides are selected such that itpossesses an estimated free fraction in human blood of between 0.1 and50%, or between 0.15 and 10%.

In some embodiments, the apparent Kd values for serum protein by EC50shift analysis is used to provide a simple and rapid means ofquantifying the propensity of experimental compounds to bind HSA andother serum proteins. A linear relationship exists between the apparentEC₅₀ in the presence of serum protein (EC′₅₀) and the amount of serumprotein added to an in vitro assay. This relationship is defined by thebinding affinity of the compound for serum proteins, expressed asK_(d)*. This term is an experimentally determined, apparent dissociationconstant that may result from the cumulative effects of multiple,experimentally indistinguishable, binding events. The form of thisrelationship is presented here in Eq. 0.1, and its derivation can befound in Copeland et al, Biorg. Med Chem Lett. 2004, 14:2309-2312, thecontents of which are incorporated herein by reference.

$\begin{matrix}{{EC}_{50}^{\prime} = {{EC}_{50} + {P\left( \frac{n}{1 + \frac{K_{d}^{*}}{{EC}_{50}}} \right)}}} & (0.1)\end{matrix}$

A significant proportion of serum protein binding can be ascribed todrug interactions with HSA, due to the very high concentration of thisprotein in serum (35-50 g/L or 530-758 μM). To calculate the K_(d) valuefor these compounds we have assumed that the shift in EC₅₀ upon proteinaddition can be ascribed fully to the HSA present in the added serum,where P is 700 μM for 100% serum, P is 70 μM for 10% serum, etc. Wefurther make the simplifying assumption that all of the compounds bindHSA with a 1:1 stoichiometry, so that the term n in Eq. (0.1) is fixedat unity. With these parameters in place we calculate the K_(d)* valuefor each stapled peptide from the changes in EC₅₀ values with increasingserum (and serum protein) concentrations by nonlinear regressionanalysis of Eq. 1.1 using Mathematica 4.1 (Wolfram Research, Inc.,www.wolfram.com). The free fraction in blood is estimated per thefollowing equation, where [HSA]_(total) is set at 700 μM, as derived byTrainor, Expert Opin. Drug Disc., 2007, 2(1):51-64, the contents ofwhich are incorporated herein by reference.

$\begin{matrix}{{FreeFraction} = \frac{K_{d}^{*}}{K_{d}^{*} + \lbrack{HSA}\rbrack_{total}}} & (0.2)\end{matrix}$

In one embodiment, the improved biological activity is measured asincreased cell penetrability or an increased ability to induceapoptosis. In yet other embodiments, the biological activity is measuredas the percentage of the number of cells killed in an in vitro assay inwhich cultured cells are exposed to an effective concentration of saidpolypeptide.

In some embodiments, the improved cross-linked polypeptide possesses anapparent affinity to human serum proteins of 1 micromolar or weaker. Inanother embodiment, the improved cross-linked polypeptide possesses anapparent affinity to human serum proteins of 3 micromolar or weaker. Inanother embodiment, the improved cross-linked polypeptide possesses anapparent affinity to human serum proteins of 10 micromolar or weaker. Inanother embodiment, the improved cross-linked polypeptide possesses anapparent affinity to human serum proteins of 70 micromolar or weaker. Inanother embodiment, the improved cross-linked polypeptide possesses anapparent affinity to human serum proteins of between 1-70 micromolar. Inanother embodiment, the improved cross-linked polypeptide possesses anapparent affinity to human serum proteins of between 1-700 micromolar.

In some embodiments, the improved cross-linked polypeptide possesses anestimated free fraction in whole blood of between 0.1-50%. In anotherembodiment, the improved cross-linked polypeptide possesses an estimatedfree fraction in whole blood of between 0.5-10%.

Crosslinked Polypeptides of the Invention

Any protein or polypeptide with a known primary amino acid sequencewhich contains a secondary structure believed to impart biologicalactivity by interaction with an intracellular protein, protein domain ornucleic acid target(s) is the subject of the present invention. Forexample, the sequence of the polypeptide can be analyzed and amino acidanalogs containing groups reactive with macrocyclization reagents can besubstituted at the appropriate positions. The appropriate positions aredetermined by ascertaining which molecular surface(s) of the secondarystructure is (are) required for biological activity and, therefore,across which other surface(s) the macrocycle forming linkers of theinvention can form a macrocycle without sterically blocking thesurface(s) required for biological activity. Such determinations aremade using methods such as X-ray crystallography of complexes betweenthe secondary structure and a natural binding partner to visualizeresidues (and surfaces) critical for activity; by sequential mutagenesisof residues in the secondary structure to functionally identify residues(and surfaces) critical for activity; or by other methods. By suchdeterminations, the appropriate amino acids are substituted with theamino acids analogs and macrocycle-forming linkers of the invention. Forexample, for an α-helical secondary structure, one surface of the helix(e.g., a molecular surface extending longitudinally along the axis ofthe helix and radially 45-135° about the axis of the helix) may berequired to make contact with another biomolecule in vivo or in vitrofor biological activity. In such a case, a macrocycle-forming linker isdesigned to link two α-carbons of the helix while extendinglongitudinally along the surface of the helix in the portion of thatsurface not directly required for activity.

In some embodiments of the invention, the peptide sequence is derivedfrom the BCL-2 family of proteins. The BCL-2 family is defined by thepresence of up to four conserved BCL-2 homology (BH) domains designatedBH1, BH2, BH3, and BH4, all of which include α-helical segments(Chittenden et al. (1995), EMBO 14:5589; Wang et al. (1996), Genes Dev.10:2859). Anti-apoptotic proteins, such as BCL-2 and BCL-X_(L), displaysequence conservation in all BH domains. Pro-apoptotic proteins aredivided into “multidomain” family members (e.g., BAK, BAX), whichpossess homology in the BH1, BH2, and BH3 domains, and “BH3-domain only”family members (e.g., BID, BAD, BIM, BIK, NOXA, PUMA), that containsequence homology exclusively in the BH3 amphipathic α-helical segment.BCL-2 family members have the capacity to form homo- and heterodimers,suggesting that competitive binding and the ratio between pro- andanti-apoptotic protein levels dictates susceptibility to death stimuli.Anti-apoptotic proteins function to protect cells from pro-apoptoticexcess, i.e., excessive programmed cell death. Additional “security”measures include regulating transcription of pro-apoptotic proteins andmaintaining them as inactive conformers, requiring either proteolyticactivation, dephosphorylation, or ligand-induced conformational changeto activate pro-death functions. In certain cell types, death signalsreceived at the plasma membrane trigger apoptosis via a mitochondrialpathway. The mitochondria can serve as a gatekeeper of cell death bysequestering cytochrome c, a critical component of a cytosolic complexwhich activates caspase 9, leading to fatal downstream proteolyticevents. Multidomain proteins such as BCL-2/BCL-X_(L) and BAK/BAX playdueling roles of guardian and executioner at the mitochondrial membrane,with their activities further regulated by upstream BH3-only members ofthe BCL-2 family. For example, BID is a member of the BH3-domain onlyfamily of pro-apoptotic proteins, and transmits death signals receivedat the plasma membrane to effector pro-apoptotic proteins at themitochondrial membrane. BID has the capability of interacting with bothpro- and anti-apoptotic proteins, and upon activation by caspase 8,triggers cytochrome c release and mitochondrial apoptosis. Deletion andmutagenesis studies determined that the amphipathic α-helical BH3segment of pro-apoptotic family members may function as a death domainand thus may represent a critical structural motif for interacting withmultidomain apoptotic proteins. Structural studies have shown that theBH3 helix can interact with anti-apoptotic proteins by inserting into ahydrophobic groove formed by the interface of BH1, 2 and 3 domains.Activated BID can be bound and sequestered by anti-apoptotic proteins(e.g., BCL-2 and BCL-X_(L)) and can trigger activation of thepro-apoptotic proteins BAX and BAK, leading to cytochrome c release anda mitochondrial apoptosis program. BAD is also a BH3-domain onlypro-apoptotic family member whose expression triggers the activation ofBAX/BAK. In contrast to BID, however, BAD displays preferential bindingto anti-apoptotic family members, BCL-2 and BCL-X_(L). Whereas the BADBH3 domain exhibits high affinity binding to BCL-2, BAD BH3 peptide isunable to activate cytochrome c release from mitochondria in vitro,suggesting that BAD is not a direct activator of BAX/BAK. Mitochondriathat over-express BCL-2 are resistant to BID-induced cytochrome crelease, but co-treatment with BAD can restore BID sensitivity.Induction of mitochondrial apoptosis by BAD appears to result fromeither: (1) displacement of BAX/BAK activators, such as BID and BID-likeproteins, from the BCL-2/BCL-XL binding pocket, or (2) selectiveoccupation of the BCL-2/BCL-XL binding pocket by BAD to preventsequestration of BID-like proteins by anti-apoptotic proteins. Thus, twoclasses of BH3-domain only proteins have emerged, BID-like proteins thatdirectly activate mitochondrial apoptosis, and BAD-like proteins, thathave the capacity to sensitize mitochondria to BID-like pro-apoptoticsby occupying the binding pockets of multidomain anti-apoptotic proteins.Various α-helical domains of BCL-2 family member proteins amendable tothe methodology disclosed herein have been disclosed (Walensky et al.(2004), Science 305:1466; and Walensky et al., U.S. Patent PublicationNo. 2005/0250680, the entire disclosures of which are incorporatedherein by reference).

In other embodiments, the peptide sequence is derived from the tumorsuppressor p53 protein which binds to the oncogene protein MDM2. TheMDM2 binding site is localized within a region of the p53 tumorsuppressor that forms an α helix. In U.S. Pat. No. 7,083,983, the entirecontents of which are incorporated herein by reference, Lane et al.disclose that the region of p53 responsible for binding to MDM2 isrepresented approximately by amino acids 13-31 (PLSQETFSDLWKLLPENNV (SEQID NO: 1)) of mature human P53 protein. Other modified sequencesdisclosed by Lane are also contemplated in the instant invention.Furthermore, the interaction of p53 and MDM2 has been discussed by Shairet al. (1997), Chem. & Biol. 4:791, the entire contents of which areincorporated herein by reference, and mutations in the p53 gene havebeen identified in virtually half of all reported cancer cases. Asstresses are imposed on a cell, p53 is believed to orchestrate aresponse that leads to either cell-cycle arrest and DNA repair, orprogrammed cell death. As well as mutations in the p53 gene that alterthe function of the p53 protein directly, p53 can be altered by changesin MDM2. The MDM2 protein has been shown to bind to p53 and disrupttranscriptional activation by associating with the transactivationdomain of p53. For example, an 11 amino-acid peptide derived from thetransactivation domain of p53 forms an amphipathic α-helix of 2.5 turnsthat inserts into the MDM2 crevice. Thus, in some embodiments, novelα-helix structures generated by the method of the present invention areengineered to generate structures that bind tightly to the helixacceptor and disrupt native protein-protein interactions. Thesestructures are then screened using high throughput techniques toidentify optimal small molecule peptides. The novel structures thatdisrupt the MDM2 interaction are useful for many applications,including, but not limited to, control of soft tissue sarcomas (whichover-expresses MDM2 in the presence of wild type p53). These cancers arethen, in some embodiments, held in check with small molecules thatintercept MDM2, thereby preventing suppression of p53. Additionally, insome embodiments, small molecules disrupters of MDM2-p53 interactionsare used as adjuvant therapy to help control and modulate the extent ofthe p53 dependent apoptosis response in conventional chemotherapy.

A non-limiting exemplary list of suitable peptide sequences for use as astarting point for optimization in accordance with the present inventionis given below:

TABLE 1 Sequence SEQ ID Cross-linked Sequence SEQ ID Name (bold =critical residues) NO: ( X  = x-link residue) NO: BH3 peptides BID-BH3QEDIIRNIARHLAQVGDSMDRSIPP 2 QEDIIRNIARHLA X VGD X MDRSIPP 25 BIM-BH3DNRPEIWIAQELRRIGDEFNAYYAR 3 DNRPEIWIAQELR X IGD X FNAYYAR 26 BAD-BH3NLWAAQRYGRELRRMSDEFVDSFKK 4 NLWAAQRYGRELR X MSD X FVDSFKK 27 PUMA-BH3EEQWAREIGAQLRRMADDLNAQYER 5 EEQWAREIGAQLR X MAD X LNAQYER 28 Hrk-BH3RSSAAQLTAARLKALGDELHQRTM 6 RSSAAQLTAARLK X LGD X LHQRTM 29 NOXAA-BH3AELPPEFAAQLRKIGDKVYCTW 7 AELPPEFAAQLR X IGD X VYCTW 30 NOXAB-BH3VPADLKDECAQLRRIGDKVNLRQKL 8 VPADLKDECAQLR X IGD X VNLRQKL 31 BMF-BH3QHRAEVQIARKLQCIADQFHRLHT 9 QHRAEVQIARKLQ X IAD X FHRLHT 32 BLK-BH3SSAAQLTAARLKALGDELHQRT 10 SSAAQLTAARLK X LGD X LHQRT 33 BIK-BH3CMEGSDALALRLACIGDEMDVSLRA 11 CMEGSDALALRLA X IGD X MDVSLRA 34 Bnip3DIERRKEVESILKKNSDWIWDWSS 12 DIERRKEVESILK X NSD X IWDWSS 35 BOK-BH3GRLAEVCAVLLRLGDELEMIRP 13 GRLAEVCAVLL X LGD X LEMIRP 36 BAX-BH3PQDASTKKSECLKRIGDELDSNMEL 14 PQDASTKKSECLK X IGD X LDSNMEL 37 BAK-BH3PSSTMGQVGRQLAIIGDDINRR 15 PSSTMGQVGRQLA X IGD X INRR 38 BCL2L1-BH3KQALREAGDEFELR 16 KQALR X AGD X FELR 39 BCL2-BH3 LSPPVVHLALALRQAGDDFSRR17 LSPPVVHLALALR X AGD X FSRR 40 BCL-XL-BH3 EVIPMAAVKQALREAGDEFELRY 18EVIPMAAVKQALR X AGD X FELRY 41 BCL-W-BH3 PADPLHQAMRAAGDEFETRF 19PADPLHQAMR X AGD X FETRF 42 MCL1-BH3 ATSRKLETLRRVGDGVQRNHETA 20ATSRKLETLR X VGD X VQRNHETA 43 MTD-BH3 LAEVCTVLLRLGDELEQIR 21 LAEVCTVLLX LGD X LEQIR 44 MAP-1-BH3 MTVGELSRALGHENGSLDP 22 MTVGELSRALG X ENG XLDP 45 NIX-BH3 VVEGEKEVEALKKSADWVSDWS 23 VVEGEKEVEALK X SAD X VSDWS 464ICD(ERBB4)-BH3 SMARDPQRYLVIQGDDRMKL 24 SMARDPQRYLV X QGD X RMKL 47Table 1 lists human sequences which target the BH3 binding site and areimplicated in cancers, autoimmune disorders, metabolic diseases andother human disease conditions.

TABLE 2 Sequence SEQ ID Cross-linked Sequence SEQ ID Name (bold =critical residues) NO: ( X  = x-link residue) NO: BH3 peptides BID-BH3QEDIIRNIARHLAQVGDSMDRSIPP 2 QEDIIRNI X RHL X QVGDSMDRSIPP 48 BIM-BH3DNRPEIWIAQELRRIGDEFNAYYAR 3 DNRPEIWI X QEL X RIGDEFNAYYAR 49 BAD-BH3NLWAAQRYGRELRRMSDEFVDSFKK 4 LWAAQRY X REL X RMSDEFVDSFKK 50 PUMA-BH3EEQWAREIGAQLRRMADDLNAQYER 5 EEQWAREI X AQL X RMADDLNAQYER 51 Hrk-BH3RSSAAQLTAARLKALGDELHQRTM 6 RSSAAQLT X ARL X ALGDELHQRTM 52 NOXAA-BH3AELPPEFAAQLRKIGDKVYCTW 7 AELPPEF X AQL X KIGDKVYCTW 53 NOXAB-BH3VPADLKDECAQLRRIGDKVNLRQKL 8 VPADLKDE X AQL X RIGDKVNLRQKL 54 BMF-BH3QHRAEVQIARKLQCIADQFHRLHT 9 QHRAEVQI X RKL X CIADQFHRLHT 55 BLK-BH3SSAAQLTAARLKALGDELHQRT 10 SSAAQLT X ARL X ALGDELHQRT 56 BIK-BH3CMEGSDALALRLACIGDEMDVSLRA 11 CMEGSDAL X LRL X CIGDEMDVSLRA 57 Bnip3DIERRKEVESILKKNSDWIWDWSS 12 DIERRKEV X SIL X KNSDWIWDWSS 58 BOK-BH3GRLAEVCAVLLRLGDELEMIRP 13 GRLAEV X AVL X RLGDELEMIRP 59 BAX-BH3PQDASTKKSECLKRIGDELDSNMEL 14 PQDASTKK X ECL X RIGDELDSNMEL 60 BAK-BH3PSSTMGQVGRQLAIIGDDINRR 15 PSSTMGQV X RQL X IIGDDINRR 61 BCL2L1-BH3KQALREAGDEFELR 16 X QAL X EAGDEFELR 62 BCL2-BH3 LSPPVVHLALALRQAGDDFSRR17 LSPPVVHL X LAL X QAGDDFSRR 63 BCL-XL-BH3 EVIPMAAVKQALREAGDEFELRY 18EVIPMAAV X QAL X EAGDEFELRY 64 BCL-W-BH3 PADPLHQAMRAAGDEFETRF 19 PADPL XQAM X AAGDEFETRF 65 MCL1-BH3 ATSRKLETLRRVGDGVQRNHETA 20 ATSRK X ETL XRVGDGVQRNHETA 66 MTD-BH3 LAEVCTVLLRLGDELEQIR 21 LAEV X TVL X RLGDELEQIR67 MAP-1-BH3 MTVGELSRALGHENGSLDP 22 MTVGEL X RAL X HENGSLDP 68 NIX-BH3VVEGEKEVEALKKSADWVSDWS 23 VVEGEKE X EAL X KSADWVSDWS 69 4ICD(ERBB4)-BH3SMARDPQRYLVIQGDDRMKL 24 SMARDP X RYL X IQGDDRMKL 70Table 2 lists human sequences which target the BH3 binding site and areimplicated in cancers, autoimmune disorders, metabolic diseases andother human disease conditions.

TABLE 3 Cross-linked Sequence (bold = SEQ ID Sequence ( X  = SEQ ID Namecritical residues) NO: x-link residue) NO: P53 peptides hp53 peptide 1LSQETFSDLWKLLPEN 71 LSQETFSD X WKLLPE X 72 hp53 peptide 2LSQETFSDLWKLLPEN 71 LSQE X FSDLWK X LPEN 73 hp53 peptide 3LSQETFSDLWKLLPEN 71 LSQ X TFSDLW X LLPEN 74 hp53 peptide 4LSQETFSDLWKLLPEN 71 LSQETF X DLWKLL X EN 75 hp53 peptide 5LSQETFSDLWKLLPEN 71 QSQQTF X NLWRLL X QN 76Table 3 lists human sequences which target the p53 binding site ofMDM2/X and are implicated in cancers.

TABLE 4 Cross-linked Sequence (bold = SEQ ID Sequence ( X  = SEQ ID Namecritical residues) NO: x-link residue) NO: GPCR peptide ligandsAngiotensin II DRVYIHPF 77 DR X Y X HPF 83 Bombesin EQRLGNQWAVGHLM 78EQRLGN X WAVGHL X 84 Bradykinin RPPGFSPFR 79 RPP X FSPFR X 85 C5aISHKDMQLGR 80 ISHKDM X LGR X 86 C3a ARASHLGLAR 81 ARASHL X LAR X 87α-melanocyte SYSMEHFRWGKPV 82 SYSM X HFRW X KPV 88 stimulating hormone

Table 4 lists sequences which target human G protein-coupled receptorsand are implicated in numerous human disease conditions (Tyndall et al.(2005), Chem. Rev. 105:793-826).

Crosslinked Polypeptides of the Invention

In some embodiments of the method, a polypeptide of the inventioncontains one crosslink. In other embodiments of the method, saidpolypeptide contains two cross-links. In some embodiments of the method,one crosslink connects two α-carbon atoms. In other embodiments of themethod, one α-carbon atom to which one crosslink is attached issubstituted with a substituent of formula R—. In another embodiment ofthe method, two α-carbon atoms to which one crosslink is attached aresubstituted with independent substituents of formula R—. In oneembodiment of the methods of the invention, R— is alkyl. For example, R—is methyl. Alternatively, R— and any portion of one crosslink takentogether can form a cyclic structure. In another embodiment of themethod, one crosslink is formed of consecutive carbon-carbon bonds. Forexample, one crosslink may comprise at least 8, 9, 10, 11, or 12consecutive bonds. In other embodiments, one crosslink may comprise atleast 7, 8, 9, 10, or 11 carbon atoms.

In another embodiment of the method, the crosslinked polypeptidecomprises an α-helical domain of a BCL-2 family member. For example, thecrosslinked polypeptide comprises a BH3 domain. In other embodiments,the crosslinked polypeptide comprises at least 60%, 70%, 80%, 85%, 90%or 95% of any of the sequences in Tables 1, 2, 3 and 4. In someembodiments of the method, the crosslinked polypeptide penetrates cellmembranes by an energy-dependent process and binds to an intracellulartarget.

In some embodiments, said helical polypeptide contains one crosslink. Inother embodiments, said helical polypeptide contains two cross-links.

In some embodiments, one crosslink connects two α-carbon atoms. In otherembodiments, one α-carbon atom to which one crosslink is attached issubstituted with a substituent of formula R—. In another embodiment, twoα-carbon atoms to which one crosslink is attached are substituted withindependent substituents of formula R—. In one embodiment of theinvention, R— is alkyl. For example, R— is methyl. Alternatively, R— andany portion of one crosslink taken together can form a cyclic structure.In another embodiment, one crosslink is formed of consecutivecarbon-carbon bonds. For example, one crosslink may comprise at least 8,9, 10, 11, or 12 consecutive bonds. In other embodiments, one crosslinkmay comprise at least 7, 8, 9, 10, or 11 carbon atoms.

In another embodiment, the crosslinked polypeptide comprises anα-helical domain of a BCL-2 family member. For example, the crosslinkedpolypeptide comprises a BH3 domain. In other embodiments, thecrosslinked polypeptide comprises at least 60%, 70%, 80%, 85%, 90% or95% of any of the sequences in Tables 1, 2, 3 and 4. In someembodiments, the crosslinked polypeptide penetrates cell membranes by anenergy-dependent process and binds to an intracellular target.

In some embodiments, the crosslinked polypeptides of the invention havethe Formula (I):

wherein:

-   each A, C, D, and E is independently a natural or non-natural amino    acid;-   B is a natural or non-natural amino acid, amino acid analog,

[—NH-L₃-CO—], [—NH-L₃-SO₂—], or [—NH-L₃-];

-   R₁ and R₂ are independently —H, alkyl, alkenyl, alkynyl, arylalkyl,    cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl,    unsubstituted or substituted with halo-;-   R₃ is hydrogen, alkyl, alkenyl, alkynyl, arylalkyl, heteroalkyl,    cycloalkyl, heterocycloalkyl, cycloalkylalkyl, cycloaryl, or    heterocycloaryl, optionally substituted with R₅;-   L is a macrocycle-forming linker of the formula -L₁-L₂-;-   L₁ and L₂ are independently alkylene, alkenylene, alkynylene,    heteroalkylene, cycloalkylene, heterocycloalkylene, cycloarylene,    heterocycloarylene, or [—R₄—K—R₄—]_(n), each being optionally    substituted with R₅;-   each R₄ is alkylene, alkenylene, alkynylene, heteroalkylene,    cycloalkylene, heterocycloalkylene, arylene, or heteroarylene;-   each K is O, S, SO, SO₂, CO, CO₂, or CONR₃;-   each R₅ is independently halogen, alkyl, —OR₆, —N(R₆)₂, —SR₆, —SOR₆,    —SO₂R₆, —CO₂R₆, a fluorescent moiety, a radioisotope or a    therapeutic agent;-   each R₆ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl,    cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a    radioisotope or a therapeutic agent;-   R₇ is —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl,    heteroalkyl, cycloalkylalkyl, heterocycloalkyl, cycloaryl, or    heterocycloaryl, optionally substituted with R₅, or part of a cyclic    structure with a D residue;-   R₈ is —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl,    heteroalkyl, cycloalkylalkyl, heterocycloalkyl, cycloaryl, or    heterocycloaryl, optionally substituted with R₅, or part of a cyclic    structure with an E residue;-   u is an integer from 0-10;-   v is an integer from 1-1000;-   w is an integer from 1-1000;-   x is an integer from 0-10;-   y is an integer from 0-10;-   z is an integer from 0-10; and-   n is an integer from 1-5.

In one example, at least one of R₁ and R₂ is alkyl, unsubstituted orsubstituted with halo-. In another example, both R₁ and R₂ areindependently alkyl, unsubstituted or substituted with halo-. In someembodiments, at least one of R₁ and R₂ is methyl. In other embodiments,R₁ and R₂ are methyl.

In some embodiments of the invention, x+y+z is at least 3. In otherembodiments of the invention, x+y+z is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.Each occurrence of A, B, C, D or E in a macrocycle or macrocycleprecursor of the invention is independently selected. For example, asequence represented by the formula [A]_(x), when x is 3, encompassesembodiments where the amino acids are not identical, e.g. Gln-Asp-Ala aswell as embodiments where the amino acids are identical, e.g.Gln-Gln-Gln. This applies for any value of x, y, or z in the indicatedranges.

In some embodiments, the crosslinked polypeptide of the inventioncomprises a secondary structure which is an α-helix and R₈ is —H,allowing intrahelical hydrogen bonding. In some embodiments, at leastone of A, B, C, D or E is an α,α-disubstituted amino acid. In oneexample, B is an α,α-disubstituted amino acid. For instance, at leastone of A, B, C, D or E is 2-aminoisobutyric acid. In other embodiments,at least one of A, B, C, D or E is

In other embodiments, the length of the macrocycle-forming linker L asmeasured from a first Cα to a second Cα is selected to stabilize adesired secondary peptide structure, such as an α-helix formed byresidues of the crosslinked polypeptide including, but not necessarilylimited to, those between the first Cα to a second Cα.

In one embodiment, the crosslinked polypeptide of Formula (I) is:

wherein each R₁ and R₂ is independently —H, alkyl, alkenyl, alkynyl,arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, orheterocycloalkyl, unsubstituted or substituted with halo-.

In related embodiments, the crosslinked polypeptide of Formula (I) is:

In other embodiments, the peptidomimetic macrocycle of Formula (I) is acompound of any of the formulas shown below:

wherein “AA” represents any natural or non-natural amino acid side chainand

is [D]_(v), [E]_(w) as defined above, and n is an integer between 0 and20, 50, 100, 200, 300, 400 or 500. In some embodiments, n is 0. In otherembodiments, n is less than 50.

Exemplary embodiments of the macrocycle-forming linker L are shownbelow.

In some embodiments, the crosslinked polypeptides of the invention havethe Formula (II):

wherein:

-   each A, C, D, and E is independently a natural or non-natural amino    acid;-   B is a natural or non-natural amino acid, amino acid analog,

[—NH-L₃-CO—], [—NH-L₃-SO₂—], or [—NH-L₃-];

-   R₁ and R₂ are independently —H, alkyl, alkenyl, alkynyl, arylalkyl,    cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl,    unsubstituted or substituted with halo-;-   R₃ is hydrogen, alkyl, alkenyl, alkynyl, arylalkyl, heteroalkyl,    cycloalkyl, heterocycloalkyl, cycloalkylalkyl, cycloaryl, or    heterocycloaryl, optionally substituted with R₅;-   L is a macrocycle-forming linker of the formula

-   L₁, L₂ and L₃ are independently alkylene, alkenylene, alkynylene,    heteroalkylene, cycloalkylene, heterocycloalkylene, cycloarylene,    heterocycloarylene, or [—R₄—K—R₄—]_(n), each being optionally    substituted with R₅;-   each R₄ is alkylene, alkenylene, alkynylene, heteroalkylene,    cycloalkylene, heterocycloalkylene, arylene, or heteroarylene;-   each K is O, S, SO, SO₂, CO, CO₂, or CONR₃;-   each R₅ is independently halogen, alkyl, —OR₆, —N(R₆)₂, —SR₆, —SOR₆,    —SO₂R₆, —CO₂R₆, a fluorescent moiety, a radioisotope or a    therapeutic agent;-   each R₆ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl,    cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a    radioisotope or a therapeutic agent;-   R₇ is —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl,    heteroalkyl, cycloalkylalkyl, heterocycloalkyl, cycloaryl, or    heterocycloaryl, optionally substituted with R₅, or part of a cyclic    structure with a D residue;-   R₈ is —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl,    heteroalkyl, cycloalkylalkyl, heterocycloalkyl, cycloaryl, or    heterocycloaryl, optionally substituted with R₅, or part of a cyclic    structure with an E residue;-   u is an integer from 0-10;-   v is an integer from 1-1000;-   w is an integer from 1-1000;-   x is an integer from 0-10;-   y is an integer from 0-10;-   z is an integer from 0-10; and-   n is an integer from 1-5.

In one example, at least one of R₁ and R₂ is alkyl, unsubstituted orsubstituted with halo-. In another example, both R₁ and R₂ areindependently alkyl, unsubstituted or substituted with halo-. In someembodiments, at least one of R₁ and R₂ is methyl. In other embodiments,R₁ and R₂ are methyl.

In some embodiments of the invention, x+y+z is at least 3. In otherembodiments of the invention, x+y+z is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.Each occurrence of A, B, C, D or E in a macrocycle or macrocycleprecursor of the invention is independently selected. For example, asequence represented by the formula [A]_(x), when x is 3, encompassesembodiments where the amino acids are not identical, e.g. Gln-Asp-Ala aswell as embodiments where the amino acids are identical, e.g.Gln-Gln-Gln. This applies for any value of x, y, or z in the indicatedranges.

In some embodiments, the crosslinked polypeptide of the inventioncomprises a secondary structure which is an α-helix and R₈ is —H,allowing intrahelical hydrogen bonding. In some embodiments, at leastone of A, B, C, D or E is an α,α-disubstituted amino acid. In oneexample, B is an α,α-disubstituted amino acid. For instance, at leastone of A, B, C, D or E is 2-aminoisobutyric acid. In other embodiments,at least one of A, B, C, D or E is

In other embodiments, the length of the macrocycle-forming linker L asmeasured from a first Cα to a second Cα is selected to stabilize adesired secondary peptide structure, such as an α-helix formed byresidues of the crosslinked polypeptide including, but not necessarilylimited to, those between the first Cα to a second Cα.

Exemplary embodiments of the macrocycle-forming linker L are shownbelow.

In other embodiments, the invention provides crosslinked polypeptides ofFormula (III):

Formula (III)wherein:

-   each A, C, D, and E is independently a natural or non-natural amino    acid;-   B is a natural or non-natural amino acid, amino acid analog,

[—NH-L₄-CO—], [—NH-L₄-SO₂—], or [—NH-L₄-];

-   R₁ and R₂ are independently —H, alkyl, alkenyl, alkynyl, arylalkyl,    cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl,    unsubstituted or substituted with halo-;-   R₃ is hydrogen, alkyl, alkenyl, alkynyl, arylalkyl, heteroalkyl,    cycloalkyl, heterocycloalkyl, cycloalkylalkyl, cycloaryl, or    heterocycloaryl, unsubstituted or substituted with R₅;-   L₁, L₂, L₃ and L₄ are independently alkylene, alkenylene,    alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene,    cycloarylene, heterocycloarylene or [—R₄—K—R₄-]n, each being    unsubstituted or substituted with R₅;    -   K is O, S, SO, SO₂, CO, CO₂, or CONR₃;-   each R₄ is alkylene, alkenylene, alkynylene, heteroalkylene,    cycloalkylene, heterocycloalkylene, arylene, or heteroarylene;-   each R₅ is independently halogen, alkyl, —OR₆, —N(R₆)₂, —SR₆, —SOR₆,    —SO₂R₆, —CO₂R₆, a fluorescent moiety, a radioisotope or a    therapeutic agent;-   each R₆ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl,    cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a    radioisotope or a therapeutic agent;-   R₇ is —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl,    heteroalkyl, cycloalkylalkyl, heterocycloalkyl, cycloaryl, or    heterocycloaryl, unsubstituted or substituted with R₅, or part of a    cyclic structure with a D residue;-   R₈ is —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl,    heteroalkyl, cycloalkylalkyl, heterocycloalkyl, cycloaryl, or    heterocycloaryl, unsubstituted or substituted with R₅, or part of a    cyclic structure with an E residue;-   u is an integer from 0-10;-   v is an integer from 1-1000;-   w is an integer from 1-1000;-   x is an integer from 0-10;-   y is an integer from 0-10;-   z is an integer from 0-10; and-   n is an integer from 1-5.

In one example, at least one of R₁ and R₂ is alkyl, unsubstituted orsubstituted with halo-. In another example, both R₁ and R₂ areindependently alkyl, unsubstituted or substituted with halo-. In someembodiments, at least one of R₁ and R₂ is methyl. In other embodiments,R₁ and R₂ are methyl.

In some embodiments of the invention, x+y+z is at least 3. In otherembodiments of the invention, x+y+z is 3, 4, 5, 6, 7, 8, 9 or 10. Eachoccurrence of A, B, C, D or E in a macrocycle or macrocycle precursor ofthe invention is independently selected. For example, a sequencerepresented by the formula [A]_(x), when x is 3, encompasses embodimentswhere the amino acids are not identical, e.g. Gln-Asp-Ala as well asembodiments where the amino acids are identical, e.g. Gln-Gln-Gln. Thisapplies for any value of x, y, or z in the indicated ranges.

In some embodiments, the crosslinked polypeptide of the inventioncomprises a secondary structure which is an α-helix and R₈ is —H,allowing intrahelical hydrogen bonding. In some embodiments, at leastone of A, B, C, D or E is an α,α-disubstituted amino acid. In oneexample, B is an α,α-disubstituted amino acid. For instance, at leastone of A, B, C, D or E is 2-aminoisobutyric acid. In other embodiments,at least one of A, B, C, D or E is

In other embodiments, the length of the macrocycle-forming linker[-L₁-S-L₂-S-L₃-] as measured from a first Cα to a second Cα is selectedto stabilize a desired secondary peptide structure, such as an α-helixformed by residues of the crosslinked polypeptide including, but notnecessarily limited to, those between the first Cα to a second Cα.

Macrocycles or macrocycle precursors are synthesized, for example, bysolution phase or solid-phase methods, and can contain bothnaturally-occurring and non-naturally-occurring amino acids. See, forexample, Hunt, “The Non-Protein Amino Acids” in Chemistry andBiochemistry of the Amino Acids, edited by G. C. Barrett, Chapman andHall, 1985. In some embodiments, the thiol moieties are the side chainsof the amino acid residues L-cysteine, D-cysteine, α-methyl-L cysteine,α-methyl-D-cysteine, L-homocysteine, D-homocysteine,α-methyl-L-homocysteine or α-methyl-D-homocysteine. A bis-alkylatingreagent is of the general formula X-L₂-Y wherein L₂ is a linker moietyand X and Y are leaving groups that are displaced by —SH moieties toform bonds with L₂. In some embodiments, X and Y are halogens such as I,Br, or Cl.

In other embodiments, D and/or E in the compound of Formula I, II or IIIare further modified in order to facilitate cellular uptake. In someembodiments, lipidating or PEGylating a crosslinked polypeptidefacilitates cellular uptake, increases bioavailability, increases bloodcirculation, alters pharmacokinetics, decreases immunogenicity and/ordecreases the needed frequency of administration.

In other embodiments, at least one of [D] and [E] in the compound ofFormula I, II or III represents a moiety comprising an additionalmacrocycle-forming linker such that the crosslinked polypeptidecomprises at least two macrocycle-forming linkers. In a specificembodiment, a crosslinked polypeptide comprises two macrocycle-forminglinkers.

In the crosslinked polypeptides of the invention, any of themacrocycle-forming linkers described herein may be used in anycombination with any of the sequences shown in Tables 1-4 and also withany of the R— substituents indicated herein.

In some embodiments, the crosslinked polypeptide comprises at least oneα-helix motif. For example, A, B and/or C in the compound of Formula I,II or III include one or more α-helices. As a general matter, α-helicesinclude between 3 and 4 amino acid residues per turn. In someembodiments, the α-helix of the crosslinked polypeptide includes 1 to 5turns and, therefore, 3 to 20 amino acid residues. In specificembodiments, the α-helix includes 1 turn, 2 turns, 3 turns, 4 turns, or5 turns. In some embodiments, the macrocycle-forming linker stabilizesan α-helix motif included within the crosslinked polypeptide. Thus, insome embodiments, the length of the macrocycle-forming linker L from afirst Cα to a second Cα is selected to increase the stability of anα-helix. In some embodiments, the macrocycle-forming linker spans from 1turn to 5 turns of the α-helix. In some embodiments, themacrocycle-forming linker spans approximately 1 turn, 2 turns, 3 turns,4 turns, or 5 turns of the α-helix. In some embodiments, the length ofthe macrocycle-forming linker is approximately 5 Å to 9 Å per turn ofthe α-helix, or approximately 6 Å to 8 Å per turn of the α-helix. Wherethe macrocycle-forming linker spans approximately 1 turn of an α-helix,the length is equal to approximately 5 carbon-carbon bonds to 13carbon-carbon bonds, approximately 7 carbon-carbon bonds to 11carbon-carbon bonds, or approximately 9 carbon-carbon bonds. Where themacrocycle-forming linker spans approximately 2 turns of an α-helix, thelength is equal to approximately 8 carbon-carbon bonds to 16carbon-carbon bonds, approximately 10 carbon-carbon bonds to 14carbon-carbon bonds, or approximately 12 carbon-carbon bonds. Where themacrocycle-forming linker spans approximately 3 turns of an α-helix, thelength is equal to approximately 14 carbon-carbon bonds to 22carbon-carbon bonds, approximately 16 carbon-carbon bonds to 20carbon-carbon bonds, or approximately 18 carbon-carbon bonds. Where themacrocycle-forming linker spans approximately 4 turns of an α-helix, thelength is equal to approximately 20 carbon-carbon bonds to 28carbon-carbon bonds, approximately 22 carbon-carbon bonds to 26carbon-carbon bonds, or approximately 24 carbon-carbon bonds. Where themacrocycle-forming linker spans approximately 5 turns of an α-helix, thelength is equal to approximately 26 carbon-carbon bonds to 34carbon-carbon bonds, approximately 28 carbon-carbon bonds to 32carbon-carbon bonds, or approximately 30 carbon-carbon bonds. Where themacrocycle-forming linker spans approximately 1 turn of an α-helix, thelinkage contains approximately 4 atoms to 12 atoms, approximately 6atoms to 10 atoms, or approximately 8 atoms. Where themacrocycle-forming linker spans approximately 2 turns of the α-helix,the linkage contains approximately 7 atoms to 15 atoms, approximately 9atoms to 13 atoms, or approximately 11 atoms. Where themacrocycle-forming linker spans approximately 3 turns of the α-helix,the linkage contains approximately 13 atoms to 21 atoms, approximately15 atoms to 19 atoms, or approximately 17 atoms. Where themacrocycle-forming linker spans approximately 4 turns of the α-helix,the linkage contains approximately 19 atoms to 27 atoms, approximately21 atoms to 25 atoms, or approximately 23 atoms. Where themacrocycle-forming linker spans approximately 5 turns of the α-helix,the linkage contains approximately 25 atoms to 33 atoms, approximately27 atoms to 31 atoms, or approximately 29 atoms. Where themacrocycle-forming linker spans approximately 1 turn of the α-helix, theresulting macrocycle forms a ring containing approximately 17 members to25 members, approximately 19 members to 23 members, or approximately 21members. Where the macrocycle-forming linker spans approximately 2 turnsof the α-helix, the resulting macrocycle forms a ring containingapproximately 29 members to 37 members, approximately 31 members to 35members, or approximately 33 members. Where the macrocycle-forminglinker spans approximately 3 turns of the α-helix, the resultingmacrocycle forms a ring containing approximately 44 members to 52members, approximately 46 members to 50 members, or approximately 48members. Where the macrocycle-forming linker spans approximately 4 turnsof the α-helix, the resulting macrocycle forms a ring containingapproximately 59 members to 67 members, approximately 61 members to 65members, or approximately 63 members. Where the macrocycle-forminglinker spans approximately 5 turns of the α-helix, the resultingmacrocycle forms a ring containing approximately 74 members to 82members, approximately 76 members to 80 members, or approximately 78members.

In other embodiments, the invention provides crosslinked polypeptides ofFormula (IV) or (IVa):

wherein:

-   each A, C, D, and E is independently a natural or non-natural amino    acid;-   B is a natural or non-natural amino acid, amino acid analog,

[—NH-L₃-CO—], [—NH-L₃-SO₂—], or [—NH-L₃-];

-   R₁ and R₂ are independently —H, alkyl, alkenyl, alkynyl, arylalkyl,    cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl,    unsubstituted or substituted with halo-, or part of a cyclic    structure with an E residue;-   R₃ is hydrogen, alkyl, alkenyl, alkynyl, arylalkyl, heteroalkyl,    cycloalkyl, heterocycloalkyl, cycloalkylalkyl, cycloaryl, or    heterocycloaryl, optionally substituted with R₅;-   L is a macrocycle-forming linker of the formula -L₁-L₂-;-   L₁ and L₂ are independently alkylene, alkenylene, alkynylene,    heteroalkylene, cycloalkylene, heterocycloalkylene, cycloarylene,    heterocycloarylene, or [—R₄—K—R₄—]_(n), each being optionally    substituted with R₅;-   each R₄ is alkylene, alkenylene, alkynylene, heteroalkylene,    cycloalkylene, heterocycloalkylene, arylene, or heteroarylene;-   each K is O, S, SO, SO₂, CO, CO₂, or CONR₃;-   each R₅ is independently halogen, alkyl, —OR₆, —N(R₆)₂, —SR₆, —SOR₆,    —SO₂R₆, —CO₂R₆, a fluorescent moiety, a radioisotope or a    therapeutic agent;-   each R₆ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl,    cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a    radioisotope or a therapeutic agent;-   R₇ is —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl,    heteroalkyl, cycloalkylalkyl, heterocycloalkyl, cycloaryl, or    heterocycloaryl, optionally substituted with R₅;-   u is an integer from 0-10;-   v is an integer from 1-1000;-   w is an integer from 1-1000;-   x is an integer from 0-10;-   y is an integer from 0-10;-   z is an integer from 0-10; and-   n is an integer from 1-5.

In one example, at least one of R₁ and R₂ is alkyl, unsubstituted orsubstituted with halo-. In another example, both R₁ and R₂ areindependently alkyl, unsubstituted or substituted with halo-. In someembodiments, at least one of R₁ and R₂ is methyl. In other embodiments,R₁ and R₂ are methyl.

In some embodiments of the invention, x+y+z is at least 1. In someembodiments of the invention, x+y+z is at least 2. In other embodimentsof the invention, x+y+z is 3, 4, 5, 6, 7, 8, 9 or 10. Each occurrence ofA, B, C, D or E in a macrocycle or macrocycle precursor of the inventionis independently selected. For example, a sequence represented by theformula [A]_(x), when x is 3, encompasses embodiments where the aminoacids are not identical, e.g. Gln-Asp-Ala as well as embodiments wherethe amino acids are identical, e.g. Gln-Gln-Gln. This applies for anyvalue of x, y, or z in the indicated ranges.

In some embodiments, the crosslinked polypeptide of the inventioncomprises a secondary structure which is an α-helix and R₈ is —H,allowing intrahelical hydrogen bonding. In some embodiments, at leastone of A, B, C, D or E is an α,α-disubstituted amino acid. In oneexample, B is an α,α-disubstituted amino acid. For instance, at leastone of A, B, C, D or E is 2-aminoisobutyric acid. In other embodiments,at least one of A, B, C, D or E is

In other embodiments, the length of the macrocycle-forming linker L asmeasured from a first Cα to a second Cα is selected to stabilize adesired secondary peptide structure, such as an α-helix formed byresidues of the crosslinked polypeptide including, but not necessarilylimited to, those between the first Cα to a second Cα.

Exemplary embodiments of the macrocycle-forming linker L are shownbelow.

Preparation of Crosslinked Polypeptides

Crosslinked polypeptides of the invention may be prepared by any of avariety of methods known in the art. For example, any of the residuesindicated by “X” in Tables 1, 2, 3 or 4 may be substituted with aresidue capable of forming a crosslinker with a second residue in thesame molecule or a precursor of such a residue.

Various methods to effect formation of crosslinked polypeptides areknown in the art. For example, the preparation of crosslinkedpolypeptides of Formula I is described in Schafmeister et al., J. Am.Chem. Soc. 122:5891-5892 (2000); Schafmeister & Verdine, J. Am. Chem.Soc. 122:5891 (2005); Walensky et al., Science 305:1466-1470 (2004);U.S. Pat. No. 7,192,713; and PCT application WO 2008/121767. Theα,α-disubstituted amino acids and amino acid precursors disclosed in thecited references may be employed in synthesis of the crosslinkedpolypeptide precursor polypeptides. Following incorporation of suchamino acids into precursor polypeptides, the terminal olefins arereacted with a metathesis catalyst, leading to the formation of thecrosslinked polypeptide.

In other embodiments, the peptidomimetic macrocyles of the invention areof Formula IV or IVa. Methods for the preparation of such macrocyclesare described, for example, in U.S. Pat. No. 7,202,332.

In some embodiments, the synthesis of these crosslinked polypeptidesinvolves a multi-step process that features the synthesis of apeptidomimetic precursor containing an azide moiety and an alkynemoiety; followed by contacting the peptidomimetic precursor with amacrocyclization reagent to generate a triazole-linked crosslinkedpolypeptide. Macrocycles or macrocycle precursors are synthesized, forexample, by solution phase or solid-phase methods, and can contain bothnaturally-occurring and non-naturally-occurring amino acids. See, forexample, Hunt, “The Non-Protein Amino Acids” in Chemistry andBiochemistry of the Amino Acids, edited by G. C. Barrett, Chapman andHall, 1985.

In some embodiments, an azide is linked to the α-carbon of a residue andan alkyne is attached to the α-carbon of another residue. In someembodiments, the azide moieties are azido-analogs of amino acidsL-lysine, D-lysine, alpha-methyl-L-lysine, alpha-methyl-D-lysine,L-ornithine, D-ornithine, alpha-methyl-L-ornithine oralpha-methyl-D-ornithine. In another embodiment, the alkyne moiety isL-propargylglycine. In yet other embodiments, the alkyne moiety is anamino acid selected from the group consisting of L-propargylglycine,D-propargylglycine, (S)-2-amino-2-methyl-4-pentynoic acid,(R)-2-amino-2-methyl-4-pentynoic acid, (S)-2-amino-2-methyl-5-hexynoicacid, (R)-2-amino-2-methyl-5-hexynoic acid,(S)-2-amino-2-methyl-6-heptynoic acid, (R)-2-amino-2-methyl-6-heptynoicacid, (S)-2-amino-2-methyl-7-octynoic acid,(R)-2-amino-2-methyl-7-octynoic acid, (S)-2-amino-2-methyl-8-nonynoicacid and (R)-2-amino-2-methyl-8-nonynoic acid.

In some embodiments, the invention provides a method for synthesizing acrosslinked polypeptide, the method comprising the steps of contacting apeptidomimetic precursor of Formula V or Formula VI:

with a macrocyclization reagent;

-   wherein v, w, x, y, z, A, B, C, D, E, R₁, R₂, R₇, R₈, L₁ and L₂ are    as defined for Formula (II); R₁₂ is —H when the macrocyclization    reagent is a Cu reagent and R₁₂ is —H or alkyl when the    macrocyclization reagent is a Ru reagent; and further wherein said    contacting step results in a covalent linkage being formed between    the alkyne and azide moiety in Formula III or Formula IV. For    example, R₁₂ may be methyl when the macrocyclization reagent is a Ru    reagent.

In the crosslinked polypeptides of the invention, at least one of R₁ andR₂ is alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl,heteroalkyl, or heterocycloalkyl, unsubstituted or substituted withhalo-. In some embodiments, both R₁ and R₂ are independently alkyl,alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl,or heterocycloalkyl, unsubstituted or substituted with halo-. In someembodiments, at least one of A, B, C, D or E is an α,α-disubstitutedamino acid. In one example, B is an α,α-disubstituted amino acid. Forinstance, at least one of A, B, C, D or E is 2-aminoisobutyric acid.

For example, at least one of R₁ and R₂ is alkyl, unsubstituted orsubstituted with halo-. In another example, both R₁ and R₂ areindependently alkyl, unsubstituted or substituted with halo-. In someembodiments, at least one of R₁ and R₂ is methyl. In other embodiments,R₁ and R₂ are methyl. The macrocyclization reagent may be a Cu reagentor a Ru reagent.

In some embodiments, the peptidomimetic precursor is purified prior tothe contacting step. In other embodiments, the crosslinked polypeptideis purified after the contacting step. In still other embodiments, thecrosslinked polypeptide is refolded after the contacting step. Themethod may be performed in solution, or, alternatively, the method maybe performed on a solid support.

Also envisioned herein is performing the method of the invention in thepresence of a target macromolecule that binds to the peptidomimeticprecursor or crosslinked polypeptide under conditions that favor saidbinding. In some embodiments, the method is performed in the presence ofa target macromolecule that binds preferentially to the peptidomimeticprecursor or crosslinked polypeptide under conditions that favor saidbinding. The method may also be applied to synthesize a library ofcrosslinked polypeptides.

In some embodiments, the alkyne moiety of the peptidomimetic precursorof Formula V or Formula VI is a sidechain of an amino acid selected fromthe group consisting of L-propargylglycine, D-propargylglycine,(S)-2-amino-2-methyl-4-pentynoic acid, (R)-2-amino-2-methyl-4-pentynoicacid, (S)-2-amino-2-methyl-5-hexynoic acid,(R)-2-amino-2-methyl-5-hexynoic acid, (S)-2-amino-2-methyl-6-heptynoicacid, (R)-2-amino-2-methyl-6-heptynoic acid,(S)-2-amino-2-methyl-7-octynoic acid, (R)-2-amino-2-methyl-7-octynoicacid, (S)-2-amino-2-methyl-8-nonynoic acid, and(R)-2-amino-2-methyl-8-nonynoic acid. In other embodiments, the azidemoiety of the peptidomimetic precursor of Formula V or Formula VI is asidechain of an amino acid selected from the group consisting ofe-azido-L-lysine, e-azido-D-lysine, e-azido-α-methyl-L-lysine,e-azido-α-methyl-D-lysine, d-azido-α-methyl-L-ornithine, andd-azido-α-methyl-D-ornithine.

In some embodiments, x+y+z is 3, and A, B and C are independentlynatural or non-natural amino acids. In other embodiments, x+y+z is 6,and A, B and C are independently natural or non-natural amino acids.

In some embodiments of peptidomimetic macrocycles of the invention,[D]_(v) and/or [E]_(w) comprise additional peptidomimetic macrocycles ormacrocyclic structures. For example, [D]_(v) may have the formula:

wherein each A, C, D′, and E′ is independently a natural or non-naturalamino acid;

B is a natural or non-natural amino acid, amino acid analog,

[—NH-L₃-CO—], [—NH-L₃-SO₂—], or [—NH-L₃-];

R₁ and R₂ are independently —H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl,unsubstituted or substituted with halo-, or part of a cyclic structurewith an E residue;

R₃ is hydrogen, alkyl, alkenyl, alkynyl, arylalkyl, heteroalkyl,cycloalkyl, heterocycloalkyl, cycloalkylalkyl, cycloaryl, orheterocycloaryl, optionally substituted with R₅;

L₁ and L₂ are independently alkylene, alkenylene, alkynylene,heteroalkylene, cycloalkylene, heterocycloalkylene, cycloarylene,heterocycloarylene, or [—R₄—K—R₄—]_(n), each being optionallysubstituted with R₅;

each R₄ is alkylene, alkenylene, alkynylene, heteroalkylene,cycloalkylene, heterocycloalkylene, arylene, or heteroarylene;

each K is O, S, SO, SO₂, CO, CO₂, or CONR₃;

each R₅ is independently halogen, alkyl, —OR₆, —N(R₆)₂, —SR₆, —SOR₆,—SO₂R₆, —CO₂R₆, a fluorescent moiety, a radioisotope or a therapeuticagent;

each R₆ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a radioisotopeor a therapeutic agent;

R₇ is —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl,cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl,optionally substituted with R₅;

v is an integer from 1-1000;

w is an integer from 1-1000; and

x is an integer from 0-10.

In another embodiment, [E]_(w) has the formula:

wherein the substituents are as defined in the preceding paragraph.

In some embodiments, the contacting step is performed in a solventselected from the group consisting of protic solvent, aqueous solvent,organic solvent, and mixtures thereof. For example, the solvent may bechosen from the group consisting of H₂O, THF, THF/H₂O, tBuOH/H₂O, DMF,DIPEA, CH₃CN or CH₂Cl₂, ClCH₂CH₂Cl or a mixture thereof. The solvent maybe a solvent which favors helix formation.

Alternative but equivalent protecting groups, leaving groups or reagentsare substituted, and certain of the synthetic steps are performed inalternative sequences or orders to produce the desired compounds.Synthetic chemistry transformations and protecting group methodologies(protection and deprotection) useful in synthesizing the compoundsdescribed herein include, for example, those such as described inLarock, Comprehensive Organic Transformations, VCH Publishers (1989);Greene and Wuts, Protective Groups in Organic Synthesis, 2d. Ed., JohnWiley and Sons (1991); Fieser and Fieser, Fieser and Fieser's Reagentsfor Organic Synthesis, John Wiley and Sons (1994); and Paquette, ed.,Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons(1995), and subsequent editions thereof.

The crosslinked polypeptides of the invention are made, for example, bychemical synthesis methods, such as described in Fields et al., Chapter3 in Synthetic Peptides: A User's Guide, ed. Grant, W. H. Freeman & Co.,New York, N.Y., 1992, p. 77. Hence, for example, peptides aresynthesized using the automated Merrifield techniques of solid phasesynthesis with the amine protected by either tBoc or Fmoc chemistryusing side chain protected amino acids on, for example, an automatedpeptide synthesizer (e.g., Applied Biosystems (Foster City, Calif.),Model 430A, 431, or 433).

One manner of producing the peptidomimetic precursors and crosslinkedpolypeptides described herein uses solid phase peptide synthesis (SPPS).The C-terminal amino acid is attached to a cross-linked polystyreneresin via an acid labile bond with a linker molecule. This resin isinsoluble in the solvents used for synthesis, making it relativelysimple and fast to wash away excess reagents and by-products. TheN-terminus is protected with the Fmoc group, which is stable in acid,but removable by base. Side chain functional groups are protected asnecessary with base stable, acid labile groups.

Longer peptidomimetic precursors are produced, for example, byconjoining individual synthetic peptides using native chemical ligation.Alternatively, the longer synthetic peptides are biosynthesized by wellknown recombinant DNA and protein expression techniques. Such techniquesare provided in well-known standard manuals with detailed protocols. Toconstruct a gene encoding a peptidomimetic precursor of this invention,the amino acid sequence is reverse translated to obtain a nucleic acidsequence encoding the amino acid sequence, preferably with codons thatare optimum for the organism in which the gene is to be expressed. Next,a synthetic gene is made, typically by synthesizing oligonucleotideswhich encode the peptide and any regulatory elements, if necessary. Thesynthetic gene is inserted in a suitable cloning vector and transfectedinto a host cell. The peptide is then expressed under suitableconditions appropriate for the selected expression system and host. Thepeptide is purified and characterized by standard methods.

The peptidomimetic precursors are made, for example, in ahigh-throughput, combinatorial fashion using, for example, ahigh-throughput polychannel combinatorial synthesizer (e.g., ThuramedTETRAS multichannel peptide synthesizer from CreoSalus, Louisville, Ky.or Model Apex 396 multichannel peptide synthesizer from AAPPTEC, Inc.,Louisville, Ky.).

The following synthetic schemes are provided solely to illustrate thepresent invention and are not intended to limit the scope of theinvention, as described herein. To simplify the drawings, theillustrative schemes depict azido amino acid analogse-azido-α-methyl-L-lysine and e-azido-α-methyl-D-lysine, and alkyneamino acid analogs L-propargylglycine, (S)-2-amino-2-methyl-4-pentynoicacid, and (S)-2-amino-2-methyl-6-heptynoic acid. Thus, in the followingsynthetic schemes, each R₁, R₂, R₇ and R₈ is —H; each L₁ is —(CH₂)₄—;and each L₂ is —(CH₂)—. However, as noted throughout the detaileddescription above, many other amino acid analogs can be employed inwhich R₁, R₂, R₇, R₈, L₁ and L₂ can be independently selected from thevarious structures disclosed herein.

Synthetic Scheme 1 describes the preparation of several compounds of theinvention. Ni(II) complexes of Schiff bases derived from the chiralauxiliary (S)-2-[N—(N′-benzylprolyl)amino]benzophenone (BPB) and aminoacids such as glycine or alanine are prepared as described in Belokon etal. (1998), Tetrahedron Asymm. 9:4249-4252. The resulting complexes aresubsequently reacted with alkylating reagents comprising an azido oralkynyl moiety to yield enantiomerically enriched compounds of theinvention. If desired, the resulting compounds can be protected for usein peptide synthesis.

In the general method for the synthesis of crosslinked polypeptidesshown in Synthetic Scheme 2, the peptidomimetic precursor contains anazide moiety and an alkyne moiety and is synthesized by solution-phaseor solid-phase peptide synthesis (SPPS) using the commercially availableamino acid N-α-Fmoc-L-propargylglycine and the N-α-Fmoc-protected formsof the amino acids (S)-2-amino-2-methyl-4-pentynoic acid,(S)-2-amino-6-heptynoic acid, (S)-2-amino-2-methyl-6-heptynoic acid,N-methyl-e-azido-L-lysine, and N-methyl-e-azido-D-lysine. Thepeptidomimetic precursor is then deprotected and cleaved from thesolid-phase resin by standard conditions (e.g., strong acid such as 95%TFA). The peptidomimetic precursor is reacted as a crude mixture or ispurified prior to reaction with a macrocyclization reagent such as aCu(I) in organic or aqueous solutions (Rostovtsev et al. (2002), Angew.Chem. Int. Ed. 41:2596-2599; Tornoe et al. (2002), J. Org. Chem.67:3057-3064; Deiters et al. (2003), J. Am. Chem. Soc. 125:11782-11783;Punna et al. (2005), Angew. Chem. Int. Ed. 44:2215-2220). In oneembodiment, the triazole forming reaction is performed under conditionsthat favor α-helix formation. In one embodiment, the macrocyclizationstep is performed in a solvent chosen from the group consisting of H₂O,THF, CH₃CN, DMF, DIPEA, tBuOH or a mixture thereof. In anotherembodiment, the macrocyclization step is performed in DMF. In someembodiments, the macrocyclization step is performed in a bufferedaqueous or partially aqueous solvent.

In the general method for the synthesis of crosslinked polypeptidesshown in Synthetic Scheme 3, the peptidomimetic precursor contains anazide moiety and an alkyne moiety and is synthesized by solid-phasepeptide synthesis (SPPS) using the commercially available amino acidN-α-Fmoc-L-propargylglycine and the N-α-Fmoc-protected forms of theamino acids (S)-2-amino-2-methyl-4-pentynoic acid,(S)-2-amino-6-heptynoic acid, (S)-2-amino-2-methyl-6-heptynoic acid,N-methyl-e-azido-L-lysine, and N-methyl-e-azido-D-lysine. Thepeptidomimetic precursor is reacted with a macrocyclization reagent suchas a Cu(I) reagent on the resin as a crude mixture (Rostovtsev et al.(2002), Angew. Chem. Int. Ed. 41:2596-2599; Tornoe et al. (2002), J.Org. Chem. 67:3057-3064; Deiters et al. (2003), J. Am. Chem. Soc.125:11782-11783; Punna et al. (2005), Angew. Chem. Int. Ed.44:2215-2220). The resultant triazole-containing crosslinked polypeptideis then deprotected and cleaved from the solid-phase resin by standardconditions (e.g., strong acid such as 95% TFA). In some embodiments, themacrocyclization step is performed in a solvent chosen from the groupconsisting of CH₂Cl₂, ClCH₂CH₂Cl, DMF, THF, NMP, DIPEA, 2,6-lutidine,pyridine, DMSO, H₂O or a mixture thereof. In some embodiments, themacrocyclization step is performed in a buffered aqueous or partiallyaqueous solvent.

In the general method for the synthesis of crosslinked polypeptidesshown in Synthetic Scheme 4, the peptidomimetic precursor contains anazide moiety and an alkyne moiety and is synthesized by solution-phaseor solid-phase peptide synthesis (SPPS) using the commercially availableamino acid N-α-Fmoc-L-propargylglycine and the N-α-Fmoc-protected formsof the amino acids (S)-2-amino-2-methyl-4-pentynoic acid,(S)-2-amino-6-heptynoic acid, (S)-2-amino-2-methyl-6-heptynoic acid,N-methyl-e-azido-L-lysine, and N-methyl-e-azido-D-lysine. Thepeptidomimetic precursor is then deprotected and cleaved from thesolid-phase resin by standard conditions (e.g., strong acid such as 95%TFA). The peptidomimetic precursor is reacted as a crude mixture or ispurified prior to reaction with a macrocyclization reagent such as aRu(II) reagents, for example Cp*RuCl(PPh₃)₂ or [Cp*RuCl]₄ (Rasmussen etal. (2007), Org. Lett. 9:5337-5339; Zhang et al. (2005), J. Am. Chem.Soc. 127:15998-15999). In some embodiments, the macrocyclization step isperformed in a solvent chosen from the group consisting of DMF, CH₃CNand THF.

In the general method for the synthesis of crosslinked polypeptidesshown in Synthetic Scheme 5, the peptidomimetic precursor contains anazide moiety and an alkyne moiety and is synthesized by solid-phasepeptide synthesis (SPPS) using the commercially available amino acidN-α-Fmoc-L-propargylglycine and the N-α-Fmoc-protected forms of theamino acids (S)-2-amino-2-methyl-4-pentynoic acid,(S)-2-amino-6-heptynoic acid, (S)-2-amino-2-methyl-6-heptynoic acid,N-methyl-e-azido-L-lysine, and N-methyl-e-azido-D-lysine. Thepeptidomimetic precursor is reacted with a macrocyclization reagent suchas a Ru(II) reagent on the resin as a crude mixture. For example, thereagent can be Cp*RuCl(PPh₃)₂ or [Cp*RuCl]₄ (Rasmussen et al. (2007),Org. Lett. 9:5337-5339; Zhang et al. (2005), J. Am. Chem. Soc.127:15998-15999). In some embodiments, the macrocyclization step isperformed in a solvent chosen from the group consisting of CH₂Cl₂,ClCH₂CH₂Cl, CH₃CN, DMF, and THF.

Several exemplary crosslinked polypeptides are shown in Table 5 (SEQ IDNos. 89-100, respectively, in order of appearance). “Nle” representsnorleucine and replaces a methionine residue. It is envisioned thatsimilar linkers are used to synthesize crosslinked polypeptides based onthe polypeptide sequences disclosed in Table 1 through Table 4.

TABLE 5

MW = 2464

MW = 2464

MW = 2478

MW = 2478

MW = 2492

MW = 2492

MW = 2464

MW = 2464

MW = 2478

MW = 2478

MW = 2492

MW = 2492

The present invention contemplates the use of non-naturally-occurringamino acids and amino acid analogs in the synthesis of the crosslinkedpolypeptides described herein. Any amino acid or amino acid analogamenable to the synthetic methods employed for the synthesis of stabletriazole containing crosslinked polypeptides can be used in the presentinvention. For example, L-propargylglycine is contemplated as a usefulamino acid in the present invention. However, other alkyne-containingamino acids that contain a different amino acid side chain are alsouseful in the invention. For example, L-propargylglycine contains onemethylene unit between the α-carbon of the amino acid and the alkyne ofthe amino acid side chain. The invention also contemplates the use ofamino acids with multiple methylene units between the α-carbon and thealkyne. Also, the azido-analogs of amino acids L-lysine, D-lysine,alpha-methyl-L-lysine, and alpha-methyl-D-lysine are contemplated asuseful amino acids in the present invention. However, other terminalazide amino acids that contain a different amino acid side chain arealso useful in the invention. For example, the azido-analog of L-lysinecontains four methylene units between the α-carbon of the amino acid andthe terminal azide of the amino acid side chain. The invention alsocontemplates the use of amino acids with fewer than or greater than fourmethylene units between the α-carbon and the terminal azide. Table 6shows some amino acids useful in the preparation of crosslinkedpolypeptides of the invention.

TABLE 6

Table 6 shows exemplary amino acids useful in the preparation ofcrosslinked polypeptides of the invention.

In some embodiments the amino acids and amino acid analogs are of theD-configuration. In other embodiments they are of the L-configuration.In some embodiments, some of the amino acids and amino acid analogscontained in the peptidomimetic are of the D-configuration while some ofthe amino acids and amino acid analogs are of the L-configuration. Insome embodiments the amino acid analogs are α,α-disubstituted, such asα-methyl-L-propargylglycine, α-methyl-D-propargylglycine,e-azido-alpha-methyl-L-lysine, and e-azido-alpha-methyl-D-lysine. Insome embodiments the amino acid analogs are N-alkylated, e.g.,N-methyl-L-propargylglycine, N-methyl-D-propargylglycine,N-methyl-e-azido-L-lysine, and N-methyl-e-azido-D-lysine.

In some embodiments, the —NH moiety of the amino acid is protected usinga protecting group, including without limitation -Fmoc and -Boc. Inother embodiments, the amino acid is not protected prior to synthesis ofthe crosslinked polypeptide.

In other embodiments, crosslinked polypeptides of Formula III aresynthesized. The following synthetic schemes describe the preparation ofsuch compounds. To simplify the drawings, the illustrative schemesdepict amino acid analogs derived from L- or D-cysteine, in which L₁ andL₃ are both —(CH₂)—. However, as noted throughout the detaileddescription above, many other amino acid analogs can be employed inwhich L₁ and L₃ can be independently selected from the variousstructures disclosed herein. The symbols “[AA]_(m)”, “[AA]_(n)”,“[AA]_(o)” represent a sequence of amide bond-linked moieties such asnatural or unnatural amino acids. As described previously, eachoccurrence of “AA” is independent of any other occurrence of “AA”, and aformula such as “[AA]_(m)” encompasses, for example, sequences ofnon-identical amino acids as well as sequences of identical amino acids.

In Scheme 6, the peptidomimetic precursor contains two —SH moieties andis synthesized by solid-phase peptide synthesis (SPPS) usingcommercially available N-α-Fmoc amino acids such asN-α-Fmoc-S-trityl-L-cysteine or N-α-Fmoc-S-trityl-D-cysteine.Alpha-methylated versions of D-cysteine or L-cysteine are generated byknown methods (Seebach et al. (1996), Angew. Chem. Int. Ed. Engl.35:2708-2748, and references therein) and then converted to theappropriately protected N-α-Fmoc-S-trityl monomers by known methods(“Bioorganic Chemistry: Peptides and Proteins”, Oxford University Press,New York: 1998, the entire contents of which are incorporated herein byreference). The precursor peptidomimetic is then deprotected and cleavedfrom the solid-phase resin by standard conditions (e.g., strong acidsuch as 95% TFA). The precursor peptidomimetic is reacted as a crudemixture or is purified prior to reaction with X-L₂-Y in organic oraqueous solutions. In some embodiments the alkylation reaction isperformed under dilute conditions (i.e. 0.15 mmol/L) to favormacrocyclization and to avoid polymerization. In some embodiments, thealkylation reaction is performed in organic solutions such as liquid NH₃(Mosberg et al. (1985), J. Am. Chem. Soc. 107:2986-2987; Szewczuk et al.(1992), Int. J. Peptide Protein Res. 40: 233-242), NH₃/MeOH, or NH₃/DMF(Or et al. (1991), J. Org. Chem. 56:3146-3149). In other embodiments,the alkylation is performed in an aqueous solution such as 6Mguanidinium HCL, pH 8 (Brunel et al. (2005), Chem. Commun.(20):2552-2554). In other embodiments, the solvent used for thealkylation reaction is DMF or dichloroethane.

In Scheme 7, the precursor peptidomimetic contains two or more —SHmoieties, of which two are specially protected to allow their selectivedeprotection and subsequent alkylation for macrocycle formation. Theprecursor peptidomimetic is synthesized by solid-phase peptide synthesis(SPPS) using commercially available N-α-Fmoc amino acids such asN-α-Fmoc-S-p-methoxytrityl-L-cysteine orN-α-Fmoc-S-p-methoxytrityl-D-cysteine. Alpha-methylated versions ofD-cysteine or L-cysteine are generated by known methods (Seebach et al.(1996), Angew. Chem. Int. Ed. Engl. 35:2708-2748, and referencestherein) and then converted to the appropriately protectedN-α-Fmoc-S-p-methoxytrityl monomers by known methods (BioorganicChemistry: Peptides and Proteins, Oxford University Press, New York:1998, the entire contents of which are incorporated herein byreference). The Mmt protecting groups of the peptidomimetic precursorare then selectively cleaved by standard conditions (e.g., mild acidsuch as 1% TFA in DCM). The precursor peptidomimetic is then reacted onthe resin with X-L₂-Y in an organic solution. For example, the reactiontakes place in the presence of a hindered base such asdiisopropylethylamine. In some embodiments, the alkylation reaction isperformed in organic solutions such as liquid NH₃ (Mosberg et al.(1985), J. Am. Chem. Soc. 107:2986-2987; Szewczuk et al. (1992), Int. J.Peptide Protein Res. 40:233-242), NH₃/MeOH or NH₃/DMF (Or et al. (1991),J. Org. Chem. 56:3146-3149). In other embodiments, the alkylationreaction is performed in DMF or dichloroethane. The crosslinkedpolypeptide is then deprotected and cleaved from the solid-phase resinby standard conditions (e.g., strong acid such as 95% TFA).

In Scheme 8, the peptidomimetic precursor contains two or more —SHmoieties, of which two are specially protected to allow their selectivedeprotection and subsequent alkylation for macrocycle formation. Thepeptidomimetic precursor is synthesized by solid-phase peptide synthesis(SPPS) using commercially available N-α-Fmoc amino acids such asN-α-Fmoc-S-p-methoxytrityl-L-cysteine,N-α-Fmoc-S-p-methoxytrityl-D-cysteine, N-α-Fmoc-S-S-t-butyl-L-cysteine,and N-α-Fmoc-S-S-t-butyl-D-cysteine. Alpha-methylated versions ofD-cysteine or L-cysteine are generated by known methods (Seebach et al.(1996), Angew. Chem. Int. Ed. Engl. 35:2708-2748, and referencestherein) and then converted to the appropriately protectedN-α-Fmoc-S-p-methoxytrityl or N-α-Fmoc-S-S-t-butyl monomers by knownmethods (Bioorganic Chemistry: Peptides and Proteins, Oxford UniversityPress, New York: 1998, the entire contents of which are incorporatedherein by reference). The S—S-tButyl protecting group of thepeptidomimetic precursor is selectively cleaved by known conditions(e.g., 20% 2-mercaptoethanol in DMF, reference: Galande et al. (2005),J. Comb. Chem. 7:174-177). The precursor peptidomimetic is then reactedon the resin with a molar excess of X-L₂-Y in an organic solution. Forexample, the reaction takes place in the presence of a hindered basesuch as diisopropylethylamine. The Mmt protecting group of thepeptidomimetic precursor is then selectively cleaved by standardconditions (e.g., mild acid such as 1% TFA in DCM). The peptidomimeticprecursor is then cyclized on the resin by treatment with a hinderedbase in organic solutions. In some embodiments, the alkylation reactionis performed in organic solutions such as NH₃/MeOH or NH₃/DMF (Or et al.(1991), J. Org. Chem. 56:3146-3149). The crosslinked polypeptide is thendeprotected and cleaved from the solid-phase resin by standardconditions (e.g., strong acid such as 95% TFA).

In Scheme 9, the peptidomimetic precursor contains two L-cysteinemoieties. The peptidomimetic precursor is synthesized by knownbiological expression systems in living cells or by known in vitro,cell-free, expression methods. The precursor peptidomimetic is reactedas a crude mixture or is purified prior to reaction with X-L2-Y inorganic or aqueous solutions. In some embodiments the alkylationreaction is performed under dilute conditions (i.e. 0.15 mmol/L) tofavor macrocyclization and to avoid polymerization. In some embodiments,the alkylation reaction is performed in organic solutions such as liquidNH₃ (Mosberg et al. (1985), J. Am. Chem. Soc. 107:2986-2987; Szewczuk etal. (1992), Int. J. Peptide Protein Res. 40:233-242), NH₃/MeOH, orNH₃/DMF (Or et al. (1991), J. Org. Chem. 56:3146-3149). In otherembodiments, the alkylation is performed in an aqueous solution such as6M guanidinium HCL, pH 8 (Brunel et al. (2005), Chem. Commun.(20):2552-2554). In other embodiments, the alkylation is performed inDMF or dichloroethane. In another embodiment, the alkylation isperformed in non-denaturing aqueous solutions, and in yet anotherembodiment the alkylation is performed under conditions that favorα-helical structure formation. In yet another embodiment, the alkylationis performed under conditions that favor the binding of the precursorpeptidomimetic to another protein, so as to induce the formation of thebound α-helical conformation during the alkylation.

Various embodiments for X and Y are envisioned which are suitable forreacting with thiol groups. In general, each X or Y is independently beselected from the general category shown in Table 5. For example, X andY are halides such as —Cl, —Br or —I. Any of the macrocycle-forminglinkers described herein may be used in any combination with any of thesequences shown in Tables 1-4 and also with any of the R— substituentsindicated herein.

TABLE 7 Examples of Reactive Groups Capable of Reacting with ThiolGroups and Resulting Linkages Resulting Covalent X or Y Linkageacrylamide Thioether halide (e.g. alkyl or aryl Thioether halide)sulfonate Thioether aziridine Thioether epoxide Thioether haloacetamideThioether maleimide Thioether sulfonate ester Thioether

Table 8 shows exemplary macrocycles of the invention (SEQ ID NOS101-106, respectively, in order of appearance). “N_(L)” representsnorleucine and replaces a methionine residue. It is envisioned thatsimilar linkers are used to synthesize crosslinked polypeptides based onthe polypeptide sequences disclosed in Table 1 through Table 4.

TABLE 8 Examples of Crosslinked polypeptides of the Invention

MW = 2477

MW = 2463

MW = 2525

MW = 2531

MW = 2475

MW = 2475 For the examples shown in this table, “N_(L)” representsnorleucine.

The present invention contemplates the use of both naturally-occurringand non-naturally-occurring amino acids and amino acid analogs in thesynthesis of the crosslinked polypeptides of Formula (III). Any aminoacid or amino acid analog amenable to the synthetic methods employed forthe synthesis of stable bis-sulfhydryl containing crosslinkedpolypeptides can be used in the present invention. For example, cysteineis contemplated as a useful amino acid in the present invention.However, sulfur containing amino acids other than cysteine that containa different amino acid side chain are also useful. For example, cysteinecontains one methylene unit between the α-carbon of the amino acid andthe terminal —SH of the amino acid side chain. The invention alsocontemplates the use of amino acids with multiple methylene unitsbetween the α-carbon and the terminal —SH. Non-limiting examples includeα-methyl-L-homocysteine and α-methyl-D-homocysteine. In some embodimentsthe amino acids and amino acid analogs are of the D-configuration. Inother embodiments they are of the L-configuration. In some embodiments,some of the amino acids and amino acid analogs contained in thepeptidomimetic are of the D-configuration while some of the amino acidsand amino acid analogs are of the L-configuration. In some embodimentsthe amino acid analogs are α,α-disubstituted, such asα-methyl-L-cysteine and α-methyl-D-cysteine.

The invention includes macrocycles in which macrocycle-forming linkersare used to link two or more —SH moieties in the peptidomimeticprecursors to form the crosslinked polypeptides of the invention. Asdescribed above, the macrocycle-forming linkers impart conformationalrigidity, increased metabolic stability and/or increased cellpenetrability. Furthermore, in some embodiments, the macrocycle-forminglinkages stabilize the α-helical secondary structure of thepeptidomimetic macrocyles. The macrocycle-forming linkers are of theformula X-L₂-Y, wherein both X and Y are the same or different moieties,as defined above. Both X and Y have the chemical characteristics thatallow one macrocycle-forming linker -L₂- to bis alkylate thebis-sulfhydryl containing peptidomimetic precursor. As defined above,the linker -L₂- includes alkylene, alkenylene, alkynylene,heteroalkylene, cycloalkylene, heterocycloalkylene, cycloarylene, orheterocycloarylene, or —R₄—K—R₄—, all of which can be optionallysubstituted with an R₅ group, as defined above. Furthermore, one tothree carbon atoms within the macrocycle-forming linkers -L₂-, otherthan the carbons attached to the —SH of the sulfhydryl containing aminoacid, are optionally substituted with a heteroatom such as N, S or O.

The L₂ component of the macrocycle-forming linker X-L₂-Y may be variedin length depending on, among other things, the distance between thepositions of the two amino acid analogs used to form the crosslinkedpolypeptide. Furthermore, as the lengths of L₁ and/or L₃ components ofthe macrocycle-forming linker are varied, the length of L₂ can also bevaried in order to create a linker of appropriate overall length forforming a stable crosslinked polypeptide. For example, if the amino acidanalogs used are varied by adding an additional methylene unit to eachof L₁ and L₃, the length of L₂ are decreased in length by the equivalentof approximately two methylene units to compensate for the increasedlengths of L₁ and L₃.

In some embodiments, L₂ is an alkylene group of the formula —(CH₂)_(n)—,where n is an integer between about 1 and about 15. For example, n is 1,2, 3, 4, 5, 6, 7, 8, 9 or 10. In other embodiments, L₂ is an alkenylenegroup. In still other embodiments, L₂ is an aryl group.

Table 9 shows additional embodiments of X-L₂-Y groups.

TABLE 9 Exemplary X—L₂—Y groups of the invention.

Each X and Y in this table, is, for example, independently Cl—, Br— orI—.

Additional methods of forming crosslinked polypeptides which areenvisioned as suitable to perform the present invention include thosedisclosed by Mustapa, M. Firouz Mohd et al., J. Org. Chem (2003), 68,pp. 8193-8198; Yang, Bin et al. Bioorg Med. Chem. Lett. (2004), 14, pp.1403-1406; U.S. Pat. No. 5,364,851; U.S. Pat. No. 5,446,128; U.S. Pat.No. 5,824,483; U.S. Pat. No. 6,713,280; and U.S. Pat. No. 7,202,332. Insuch embodiments, aminoacid precursors are used containing an additionalsubstituent R— at the alpha position. Such aminoacids are incorporatedinto the macrocycle precursor at the desired positions, which may be atthe positions where the crosslinker is substituted or, alternatively,elsewhere in the sequence of the macrocycle precursor. Cyclization ofthe precursor is then effected according to the indicated method.

Assays

The properties of the crosslinked polypeptides of the invention areassayed, for example, by using the methods described below.

Assay to Determine α-helicity.

In solution, the secondary structure of polypeptides with α-helicaldomains will reach a dynamic equilibrium between random coil structuresand α-helical structures, often expressed as a “percent helicity”. Thus,for example, unmodified pro-apoptotic BH3 domains are predominantlyrandom coils in solution, with α-helical content usually under 25%.Peptidomimetic macrocycles with optimized linkers, on the other hand,possess, for example, an alpha-helicity that is at least two-foldgreater than that of a corresponding uncrosslinked polypeptide. In someembodiments, macrocycles of the invention will possess an alpha-helicityof greater than 50%. To assay the helicity of peptidomimetic macrocylesof the invention, such as BH3 domain-based macrocycles, the compoundsare dissolved in an aqueous solution (e.g. 50 mM potassium phosphatesolution at pH 7, or distilled H₂O, to concentrations of 25-50 μM).Circular dichroism (CD) spectra are obtained on a spectropolarimeter(e.g., Jasco J-710) using standard measurement parameters (e.g.temperature, 20° C.; wavelength, 190-260 nm; step resolution, 0.5 nm;speed, 20 nm/sec; accumulations, 10; response, 1 sec; bandwidth, 1 nm;path length, 0.1 cm). The α-helical content of each peptide iscalculated by dividing the mean residue ellipticity (e.g. [Φ]222obs) bythe reported value for a model helical decapeptide (Yang et al. (1986),Methods Enzymol. 130:208)).

Assay to Determine Melting Temperature (Tm).

A peptidomimetic macrocycle of the invention comprising a secondarystructure such as an α-helix exhibits, for example, a higher meltingtemperature than a corresponding uncrosslinked polypeptide. Typicallypeptidomimetic macrocycles of the invention exhibit Tm of >60° C.representing a highly stable structure in aqueous solutions. To assaythe effect of macrocycle formation on meltine temperature,peptidomimetic macrocycles or unmodified peptides are dissolved indistilled H₂O (e.g. at a final concentration of 50 μM) and the Tm isdetermined by measuring the change in ellipticity over a temperaturerange (e.g. 4 to 95° C.) on a spectropolarimeter (e.g., Jasco J-710)using standard parameters (e.g. wavelength 222 nm; step resolution, 0.5nm; speed, 20 nm/sec; accumulations, 10; response, 1 sec; bandwidth, 1nm; temperature increase rate: 1° C./min; path length, 0.1 cm).

Protease Resistance Assay.

The amide bond of the peptide backbone is susceptible to hydrolysis byproteases, thereby rendering peptidic compounds vulnerable to rapiddegradation in vivo. Peptide helix formation, however, typically buriesthe amide backbone and therefore may shield it from proteolyticcleavage. The peptidomimetic macrocycles of the present invention may besubjected to in vitro trypsin proteolysis to assess for any change indegradation rate compared to a corresponding uncrosslinked polypeptide.For example, the peptidomimetic macrocycle and a correspondinguncrosslinked polypeptide are incubated with trypsin agarose and thereactions quenched at various time points by centrifugation andsubsequent HPLC injection to quantitate the residual substrate byultraviolet absorption at 280 nm. Briefly, the peptidomimetic macrocycleand peptidomimetic precursor (5 mcg) are incubated with trypsin agarose(Pierce) (S/E˜125) for 0, 10, 20, 90, and 180 minutes. Reactions arequenched by tabletop centrifugation at high speed; remaining substratein the isolated supernatant is quantified by HPLC-based peak detectionat 280 nm. The proteolytic reaction displays first order kinetics andthe rate constant, k, is determined from a plot of ln [S] versus time(k=−1×slope).

Ex Vivo Stability Assay.

Peptidomimetic macrocycles with optimized linkers possess, for example,an ex vivo half-life that is at least two-fold greater than that of acorresponding uncrosslinked polypeptide, and possess an ex vivohalf-life of 12 hours or more. For ex vivo serum stability studies, avariety of assays may be used. For example, a peptidomimetic macrocycleand/or a corresponding uncrosslinked polypeptide (2 mcg) are eachincubated with fresh mouse, rat and/or human serum (e.g. 1-2 mL) at 37°C. for 0, 1, 2, 4, 8, and 24 hours. Samples of differing macrocycleconcentration may be prepared by serial dilution with serum. Todetermine the level of intact compound, the following procedure may beused: The samples are extracted by transferring 100 μl of sera to 2 mlcentrifuge tubes followed by the addition of 10 μL of 50% formic acidand 500 μL acetonitrile and centrifugation at 14,000 RPM for 10 min at4±2° C. The supernatants are then transferred to fresh 2 ml tubes andevaporated on Turbovap under N₂<10 psi, 37° C. The samples arereconstituted in 100 μL of 50:50 acetonitrile:water and submitted toLC-MS/MS analysis. Equivalent or similar procedures for testing ex vivostability are known and may be used to determine stability ofmacrocycles in serum.

In vitro Binding Assays.

To assess the binding and affinity of peptidomimetic macrocycles andpeptidomimetic precursors to acceptor proteins, a fluorescencepolarization assay (FPA) is used, for example. The FPA techniquemeasures the molecular orientation and mobility using polarized lightand fluorescent tracer. When excited with polarized light, fluorescenttracers (e.g., FITC) attached to molecules with high apparent molecularweights (e.g. FITC-labeled peptides bound to a large protein) emithigher levels of polarized fluorescence due to their slower rates ofrotation as compared to fluorescent tracers attached to smallermolecules (e.g. FITC-labeled peptides that are free in solution).

For example, fluoresceinated peptidomimetic macrocycles (25 nM) areincubated with the acceptor protein (25-1000 nM) in binding buffer (140mM NaCl, 50 mM Tris-HCL, pH 7.4) for 30 minutes at room temperature.Binding activity is measured, for example, by fluorescence polarizationon a luminescence spectrophotometer (e.g. Perkin-Elmer LS50B). Kd valuesmay be determined by nonlinear regression analysis using, for example,Graphpad Prism software (GraphPad Software, Inc., San Diego, Calif.). Apeptidomimetic macrocycle of the invention shows, in some instances,similar or lower Kd than a corresponding uncrosslinked polypeptide.

Acceptor proteins for BH3-peptides such as BCL-2, BCL-X_(L), BAX or MCL1may, for example, be used in this assay. Acceptor proteins for p53peptides such as MDM2 or MDMX may also be used in this assay.

In vitro Displacement Assays to Characterize Antagonists ofPeptide-Protein Interactions.

To assess the binding and affinity of compounds that antagonize theinteraction between a peptide (e.g. a BH3 peptide or a p53 peptide) andan acceptor protein, a fluorescence polarization assay (FPA) utilizing afluoresceinated peptidomimetic macrocycle derived from a peptidomimeticprecursor sequence is used, for example. The FPA technique measures themolecular orientation and mobility using polarized light and fluorescenttracer. When excited with polarized light, fluorescent tracers (e.g.,FITC) attached to molecules with high apparent molecular weights (e.g.FITC-labeled peptides bound to a large protein) emit higher levels ofpolarized fluorescence due to their slower rates of rotation as comparedto fluorescent tracers attached to smaller molecules (e.g. FITC-labeledpeptides that are free in solution). A compound that antagonizes theinteraction between the fluoresceinated peptidomimetic macrocycle and anacceptor protein will be detected in a competitive binding FPAexperiment.

For example, putative antagonist compounds (1 nM to 1 mM) and afluoresceinated peptidomimetic macrocycle (25 nM) are incubated with theacceptor protein (50 nM) in binding buffer (140 mM NaCl, 50 mM Tris-HCL,pH 7.4) for 30 minutes at room temperature. Antagonist binding activityis measured, for example, by fluorescence polarization on a luminescencespectrophotometer (e.g. Perkin-Elmer LS50B). Kd values may be determinedby nonlinear regression analysis using, for example, Graphpad Prismsoftware (GraphPad Software, Inc., San Diego, Calif.).

Any class of molecule, such as small organic molecules, peptides,oligonucleotides or proteins can be examined as putative antagonists inthis assay. Acceptor proteins for BH3-peptides such as BCL2, BCL-XL, BAXor MCL1 can be used in this assay. Acceptor proteins for p53 peptidessuch as MDM2 or MDMX can be used in this assay.

Binding Assays in Intact Cells.

It is possible to measure binding of peptides or crosslinkedpolypeptides to their natural acceptors in intact cells byimmunoprecipitation experiments. For example, intact cells are incubatedwith fluoresceinated (FITC-labeled) compounds for 4-24 hrs in theabsence or presence of serum. Cells are then pelleted and incubated inlysis buffer (50 mM Tris [pH 7.6], 150 mM NaCl, 1% CHAPS and proteaseinhibitor cocktail) for 10 minutes at 4° C. Extracts are centrifuged at14,000 rpm for 15 minutes and supernatants collected and incubated with10 μl goat anti-FITC antibody for 2 hrs, rotating at 4° C. followed byfurther 2 hrs incubation at 4° C. with protein A/G Sepharose (50 μl of50% bead slurry). After quick centrifugation, the pellets are washed inlysis buffer containing increasing salt concentration (e.g., 150, 300,500 mM). The beads are then re-equilibrated at 150 mM NaCl beforeaddition of SDS-containing sample buffer and boiling. Aftercentrifugation, the supernatants are optionally electrophoresed using4%-12% gradient Bis-Tris gels followed by transfer into Immobilon-Pmembranes. After blocking, blots are optionally incubated with anantibody that detects FITC and also with one or more antibodies thatdetect proteins that bind to the crosslinked polypeptide, includingBCL2, MCL1, BCL-XL, A1, BAX, BAK, MDM2 or MDMX.

Cellular Penetrability Assays.

To measure the cell penetrability of peptides or crosslinkedpolypeptides, intact cells are incubated with fluoresceinatedcrosslinked polypeptides (100 μM) for 4 hrs in serum-free media or inmedia supplemented with human serum at 37° C., washed twice with mediaand incubated with trypsin (0.25%) for 10 min at 37° C. The cells arewashed again and resuspended in PBS. Cellular fluorescence is analyzed,for example, by using either a FACSCalibur flow cytometer or Cellomics'KINETICSCAN® HCS Reader (An automated device for analysis of cellularand intracellular spatial parameters, over time, in populations ofliving cells).

Cellular Efficacy Assays.

The efficacy of certain crosslinked polypeptides is determined, forexample, in cell-based killing assays using a variety of tumorigenic andnon-tumorigenic cell lines and primary cells derived from human or mousecell populations. Cell viability is monitored, for example, over 24-96hrs of incubation with crosslinked polypeptides (0.5 to 50 μM) toidentify those that kill at EC50<10 μM. Several standard assays thatmeasure cell viability are commercially available and are optionallyused to assess the efficacy of the crosslinked polypeptides. Inaddition, assays that measure Annexin V and caspase activation areoptionally used to assess whether the crosslinked polypeptides killcells by activating the apoptotic machinery. For example, the CellTiter-glo assay is used which determines cell viability as a function ofintracellular ATP concentration.

In Vivo Stability Assay.

To investigate the in vivo stability of crosslinked polypeptides, thecompounds are, for example, administered to mice and/or rats by IV, IP,PO or inhalation routes at concentrations ranging from 0.1 to 50 mg/kgand blood specimens withdrawn at 0′, 5′, 15′, 30′, 1 hr, 4 hrs, 8 hrsand 24 hours post-injection. Levels of intact compound in 25 μL of freshserum are then measured by LC-MS/MS as above.

In Vivo Efficacy in Animal Models.

To determine the anti-oncogenic activity of crosslinked polypeptides ofthe invention in vivo, the compounds are, for example, given alone (IP,IV, PO, by inhalation or nasal routes) or in combination withsub-optimal doses of relevant chemotherapy (e.g., cyclophosphamide,doxorubicin, etoposide). In one example, 5×10⁶ RS4; 11 cells(established from the bone marrow of a patient with acute lymphoblasticleukemia) that stably express luciferase are injected by tail vein inNOD-SCID mice 3 hrs after they have been subjected to total bodyirradiation. If left untreated, this form of leukemia is fatal in 3weeks in this model. The leukemia is readily monitored, for example, byinjecting the mice with D-luciferin (60 mg/kg) and imaging theanesthetized animals (e.g., Xenogen In Vivo Imaging System, Caliper LifeSciences, Hopkinton, Mass.). Total body bioluminescence is quantified byintegration of photonic flux (photons/sec) by Living Image Software(Caliper Life Sciences, Hopkinton, Mass.). Peptidomimetic macrocyclesalone or in combination with sub-optimal doses of relevantchemotherapeutics agents are, for example, administered to leukemic mice(10 days after injection/day 1 of experiment, in bioluminescence rangeof 14-16) by tail vein or IP routes at doses ranging from 0.1 mg/kg to50 mg/kg for 7 to 21 days. Optionally, the mice are imaged throughoutthe experiment every other day and survival monitored daily for theduration of the experiment. Expired mice are optionally subjected tonecropsy at the end of the experiment. Another animal model isimplantation into NOD-SCID mice of DoHH2, a cell line derived from humanfollicular lymphoma, that stably expresses luciferase. These in vivotests optionally generate preliminary pharmacokinetic, pharmacodynamicand toxicology data.

Clinical Trials.

To determine the suitability of the crosslinked polypeptides of theinvention for treatment of humans, clinical trials are performed. Forexample, patients diagnosed with cancer and in need of treatment areselected and separated in treatment and one or more control groups,wherein the treatment group is administered a crosslinked polypeptide ofthe invention, while the control groups receive a placebo or a knownanti-cancer drug. The treatment safety and efficacy of the crosslinkedpolypeptides of the invention can thus be evaluated by performingcomparisons of the patient groups with respect to factors such assurvival and quality-of-life. In this example, the patient group treatedwith a crosslinked polypeptide show improved long-term survival comparedto a patient control group treated with a placebo.

Pharmaceutical Compositions and Routes of Administration

The crosslinked polypeptides of the invention also includepharmaceutically acceptable derivatives or prodrugs thereof. A“pharmaceutically acceptable derivative” means any pharmaceuticallyacceptable salt, ester, salt of an ester, pro-drug or other derivativeof a compound of this invention which, upon administration to arecipient, is capable of providing (directly or indirectly) a compoundof this invention. Particularly favored pharmaceutically acceptablederivatives are those that increase the bioavailability of the compoundsof the invention when administered to a mammal (e.g., by increasingabsorption into the blood of an orally administered compound) or whichincreases delivery of the active compound to a biological compartment(e.g., the brain or lymphatic system) relative to the parent species.Some pharmaceutically acceptable derivatives include a chemical groupwhich increases aqueous solubility or active transport across thegastrointestinal mucosa.

In some embodiments, the crosslinked polypeptides of the invention aremodified by covalently or non-covalently joining appropriate functionalgroups to enhance selective biological properties. Such modificationsinclude those which increase biological penetration into a givenbiological compartment (e.g., blood, lymphatic system, central nervoussystem), increase oral availability, increase solubility to allowadministration by injection, alter metabolism, and alter rate ofexcretion.

Pharmaceutically acceptable salts of the compounds of this inventioninclude those derived from pharmaceutically acceptable inorganic andorganic acids and bases. Examples of suitable acid salts includeacetate, adipate, benzoate, benzenesulfonate, butyrate, citrate,digluconate, dodecylsulfate, formate, fumarate, glycolate, hemisulfate,heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide,lactate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate,nicotinate, nitrate, palmoate, phosphate, picrate, pivalate, propionate,salicylate, succinate, sulfate, tartrate, tosylate and undecanoate.Salts derived from appropriate bases include alkali metal (e.g.,sodium), alkaline earth metal (e.g., magnesium), ammonium and N-(alkyl)₄⁺ salts.

For preparing pharmaceutical compositions from the compounds of thepresent invention, pharmaceutically acceptable carriers include eithersolid or liquid carriers. Solid form preparations include powders,tablets, pills, capsules, cachets, suppositories, and dispersiblegranules. A solid carrier can be one or more substances, which also actsas diluents, flavoring agents, binders, preservatives, tabletdisintegrating agents, or an encapsulating material. Details ontechniques for formulation and administration are well described in thescientific and patent literature, see, e.g., the latest edition ofRemington's Pharmaceutical Sciences, Maack Publishing Co, Easton Pa.

In powders, the carrier is a finely divided solid, which is in a mixturewith the finely divided active component. In tablets, the activecomponent is mixed with the carrier having the necessary bindingproperties in suitable proportions and compacted in the shape and sizedesired.

Suitable solid excipients are carbohydrate or protein fillers include,but are not limited to sugars, including lactose, sucrose, mannitol, orsorbitol; starch from corn, wheat, rice, potato, or other plants;cellulose such as methyl cellulose, hydroxypropylmethyl-cellulose, orsodium carboxymethylcellulose; and gums including arabic and tragacanth;as well as proteins such as gelatin and collagen. If desired,disintegrating or solubilizing agents are added, such as thecross-linked polyvinyl pyrrolidone, agar, alginic acid, or a saltthereof, such as sodium alginate.

Liquid form preparations include solutions, suspensions, and emulsions,for example, water or water/propylene glycol solutions. For parenteralinjection, liquid preparations can be formulated in solution in aqueouspolyethylene glycol solution. The term “parenteral” as used hereintypically refers to modes of administration including intravenous,intraarterial, intramuscular, intraperitoneal, intrasternal, andsubcutaneous.

The pharmaceutical preparation is preferably in unit dosage form. Insuch form the preparation is subdivided into unit doses containingappropriate quantities of the active component. The unit dosage form canbe a packaged preparation, the package containing discrete quantities ofpreparation, such as packeted tablets, capsules, and powders in vials orampoules. Also, the unit dosage form can be a capsule, tablet, cachet,or lozenge itself, or it can be the appropriate number of any of thesein packaged form.

When the compositions of this invention comprise a combination of acrosslinked polypeptide and one or more additional therapeutic orprophylactic agents, both the compound and the additional agent shouldbe present at dosage levels of between about 1 to 100%, and morepreferably between about 5 to 95% of the dosage normally administered ina monotherapy regimen. In some embodiments, the additional agents areadministered separately, as part of a multiple dose regimen, from thecompounds of this invention. Alternatively, those agents are part of asingle dosage form, mixed together with the compounds of this inventionin a single composition.

Methods of administration that can be used in the present inventioninclude but are not limited to intradermal, intramuscular,intraperitoneal, intravenous, subcutaneous, intranasal, epidural, oral,sublingual, intracerebral, intravaginal, transdermal, rectal, byinhalation, or topical by application to ears, nose, eyes, or skin.

Methods of Use

In one aspect, the present invention provides novel crosslinkedpolypeptides that are useful in competitive binding assays to identifyagents which bind to the natural ligand(s) of the proteins or peptidesupon which the crosslinked polypeptides are modeled. For example, in thep53 MDM2 system, labeled stabilized crosslinked polypeptides based onthe p53 is used in an MDM2 binding assay along with small molecules thatcompetitively bind to MDM2. Competitive binding studies allow for rapidin vitro evaluation and determination of drug candidates specific forthe p53/MDM2 system. Likewise in the BH3/BCL-X_(L) anti-apoptotic systemlabeled crosslinked polypeptides based on BH3 can be used in a BCL-X_(L)binding assay along with small molecules that competitively bind toBCL-X_(L). Competitive binding studies allow for rapid in vitroevaluation and determination of drug candidates specific for theBH3/BCL-X_(L) system. The invention further provides for the generationof antibodies against the crosslinked polypeptides. In some embodiments,these antibodies specifically bind both the crosslinked polypeptides andthe p53 or BH3 crosslinked polypeptide precursors upon which thecrosslinked polypeptides are derived. Such antibodies, for example,disrupt the p53/MDM2 or BH3/BCL-XL systems, respectively.

In another aspect, the present invention provides for both prophylacticand therapeutic methods of treating a subject at risk of (or susceptibleto) a disorder or having a disorder associated with aberrant (e.g.,insufficient or excessive) BCL-2 family member expression or activity(e.g., extrinsic or intrinsic apoptotic pathway abnormalities). It isbelieved that some BCL-2 type disorders are caused, at least in part, byan abnormal level of one or more BCL-2 family members (e.g., over orunder expression), or by the presence of one or more BCL-2 familymembers exhibiting abnormal activity. As such, the reduction in thelevel and/or activity of the BCL-2 family member or the enhancement ofthe level and/or activity of the BCL-2 family member, is used, forexample, to ameliorate or reduce the adverse symptoms of the disorder.

In another aspect, the present invention provides methods for treatingor preventing hyperproliferative disease by interfering with theinteraction or binding between p53 and MDM2 in tumor cells. Thesemethods comprise administering an effective amount of a compound of theinvention to a warm blooded animal, including a human, or to tumor cellscontaining wild type p53. In some embodiments, the administration of thecompounds of the present invention induces cell growth arrest orapoptosis. In other or further embodiments, the present invention isused to treat disease and/or tumor cells comprising elevated MDM2levels. Elevated levels of MDM2 as used herein refers to MDM2 levelsgreater than those found in cells containing more than the normal copynumber (2) of mdm2 or above about 10,000 molecules of MDM2 per cell asmeasured by ELISA and similar assays (Picksley et al. (1994), Oncogene9, 2523 2529).

As used herein, the term “treatment” is defined as the application oradministration of a therapeutic agent to a patient, or application oradministration of a therapeutic agent to an isolated tissue or cell linefrom a patient, who has a disease, a symptom of disease or apredisposition toward a disease, with the purpose to cure, heal,alleviate, relieve, alter, remedy, ameliorate, improve or affect thedisease, the symptoms of disease or the predisposition toward disease.

In some embodiments, the crosslinked polypeptides of the invention areused to treat, prevent, and/or diagnose cancers and neoplasticconditions. As used herein, the terms “cancer”, “hyperproliferative” and“neoplastic” refer to cells having the capacity for autonomous growth,i.e., an abnormal state or condition characterized by rapidlyproliferating cell growth. Hyperproliferative and neoplastic diseasestates may be categorized as pathologic, i.e., characterizing orconstituting a disease state, or may be categorized as non-pathologic,i.e., a deviation from normal but not associated with a disease state.The term is meant to include all types of cancerous growths or oncogenicprocesses, metastatic tissues or malignantly transformed cells, tissues,or organs, irrespective of histopathologic type or stage ofinvasiveness. A metastatic tumor can arise from a multitude of primarytumor types, including but not limited to those of breast, lung, liver,colon and ovarian origin. “Pathologic hyperproliferative” cells occur indisease states characterized by malignant tumor growth. Examples ofnon-pathologic hyperproliferative cells include proliferation of cellsassociated with wound repair. Examples of cellular proliferative and/ordifferentiative disorders include cancer, e.g., carcinoma, sarcoma, ormetastatic disorders. In some embodiments, the peptidomimeticsmacrocycles are novel therapeutic agents for controlling breast cancer,ovarian cancer, colon cancer, lung cancer, metastasis of such cancersand the like.

Examples of cancers or neoplastic conditions include, but are notlimited to, a fibrosarcoma, myosarcoma, liposarcoma, chondrosarcoma,osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma,lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma,Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, gastric cancer,esophageal cancer, rectal cancer, pancreatic cancer, ovarian cancer,prostate cancer, uterine cancer, cancer of the head and neck, skincancer, brain cancer, squamous cell carcinoma, sebaceous glandcarcinoma, papillary carcinoma, papillary adenocarcinoma,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, testicularcancer, small cell lung carcinoma, non-small cell lung carcinoma,bladder carcinoma, epithelial carcinoma, glioma, astrocytoma,medulloblastoma, craniopharyngioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma,melanoma, neuroblastoma, retinoblastoma, leukemia, lymphoma, or Kaposisarcoma.

Examples of proliferative disorders include hematopoietic neoplasticdisorders. As used herein, the term “hematopoietic neoplastic disorders”includes diseases involving hyperplastic/neoplastic cells ofhematopoietic origin, e.g., arising from myeloid, lymphoid or erythroidlineages, or precursor cells thereof. Preferably, the diseases arisefrom poorly differentiated acute leukemias, e.g., erythroblasticleukemia and acute megakaryoblastic leukemia. Additional exemplarymyeloid disorders include, but are not limited to, acute promyeloidleukemia (APML), acute myelogenous leukemia (AML) and chronicmyelogenous leukemia (CML) (reviewed in Vaickus (1991), Crit Rev.Oncol./Hemotol. 11:267-97); lymphoid malignancies include, but are notlimited to acute lymphoblastic leukemia (ALL) which includes B-lineageALL and T-lineage ALL, chronic lymphocytic leukemia (CLL),prolymphocytic leukemia (PLL), hairy cell leukemia (HLL) andWaldenstrom's macroglobulinemia (WM). Additional forms of malignantlymphomas include, but are not limited to non-Hodgkin lymphoma andvariants thereof, peripheral T cell lymphomas, adult T cellleukemia/lymphoma (ATL), cutaneous T-cell lymphoma (CTCL), largegranular lymphocytic leukemia (LGF), Hodgkin's disease and Reed-Stembergdisease.

Examples of cellular proliferative and/or differentiative disorders ofthe breast include, but are not limited to, proliferative breast diseaseincluding, e.g., epithelial hyperplasia, sclerosing adenosis, and smallduct papillomas; tumors, e.g., stromal tumors such as fibroadenoma,phyllodes tumor, and sarcomas, and epithelial tumors such as large ductpapilloma; carcinoma of the breast including in situ (noninvasive)carcinoma that includes ductal carcinoma in situ (including Paget'sdisease) and lobular carcinoma in situ, and invasive (infiltrating)carcinoma including, but not limited to, invasive ductal carcinoma,invasive lobular carcinoma, medullary carcinoma, colloid (mucinous)carcinoma, tubular carcinoma, and invasive papillary carcinoma, andmiscellaneous malignant neoplasms. Disorders in the male breast include,but are not limited to, gynecomastia and carcinoma.

Examples of cellular proliferative and/or differentiative disorders ofthe lung include, but are not limited to, bronchogenic carcinoma,including paraneoplastic syndromes, bronchioloalveolar carcinoma,neuroendocrine tumors, such as bronchial carcinoid, miscellaneoustumors, and metastatic tumors; pathologies of the pleura, includinginflammatory pleural effusions, noninflammatory pleural effusions,pneumothorax, and pleural tumors, including solitary fibrous tumors(pleural fibroma) and malignant mesothelioma.

Examples of cellular proliferative and/or differentiative disorders ofthe colon include, but are not limited to, non-neoplastic polyps,adenomas, familial syndromes, colorectal carcinogenesis, colorectalcarcinoma, and carcinoid tumors.

Examples of cellular proliferative and/or differentiative disorders ofthe liver include, but are not limited to, nodular hyperplasias,adenomas, and malignant tumors, including primary carcinoma of the liverand metastatic tumors.

Examples of cellular proliferative and/or differentiative disorders ofthe ovary include, but are not limited to, ovarian tumors such as,tumors of coelomic epithelium, serous tumors, mucinous tumors,endometrioid tumors, clear cell adenocarcinoma, cystadenofibroma,Brenner tumor, surface epithelial tumors; germ cell tumors such asmature (benign) teratomas, monodermal teratomas, immature malignantteratomas, dysgerminoma, endodermal sinus tumor, choriocarcinoma; sexcord-stomal tumors such as, granulosa-theca cell tumors,thecomafibromas, androblastomas, hill cell tumors, and gonadoblastoma;and metastatic tumors such as Krukenberg tumors.

In other or further embodiments, the peptidomimetics macrocyclesdescribed herein are used to treat, prevent or diagnose conditionscharacterized by overactive cell death or cellular death due tophysiologic insult, etc. Some examples of conditions characterized bypremature or unwanted cell death are or alternatively unwanted orexcessive cellular proliferation include, but are not limited tohypocellular/hypoplastic, acellular/aplastic, orhypercellular/hyperplastic conditions. Some examples include hematologicdisorders including but not limited to fanconi anemia, aplastic anemia,thalaessemia, congenital neutropenia, myelodysplasia

In other or further embodiments, the crosslinked polypeptides of theinvention that act to decrease apoptosis are used to treat disordersassociated with an undesirable level of cell death. Thus, in someembodiments, the anti-apoptotic crosslinked polypeptides of theinvention are used to treat disorders such as those that lead to celldeath associated with viral infection, e.g., infection associated withinfection with human immunodeficiency virus (HIV). A wide variety ofneurological diseases are characterized by the gradual loss of specificsets of neurons, and the anti-apoptotic crosslinked polypeptides of theinvention are used, in some embodiments, in the treatment of thesedisorders. Such disorders include Alzheimer's disease, Parkinson'sdisease, amyotrophic lateral sclerosis (ALS) retinitis pigmentosa,spinal muscular atrophy, and various forms of cerebellar degeneration.The cell loss in these diseases does not induce an inflammatoryresponse, and apoptosis appears to be the mechanism of cell death. Inaddition, a number of hematologic diseases are associated with adecreased production of blood cells. These disorders include anemiaassociated with chronic disease, aplastic anemia, chronic neutropenia,and the myelodysplastic syndromes. Disorders of blood cell production,such as myelodysplastic syndrome and some forms of aplastic anemia, areassociated with increased apoptotic cell death within the bone marrow.These disorders could result from the activation of genes that promoteapoptosis, acquired deficiencies in stromal cells or hematopoieticsurvival factors, or the direct effects of toxins and mediators ofimmune responses. Two common disorders associated with cell death aremyocardial infarctions and stroke. In both disorders, cells within thecentral area of ischemia, which is produced in the event of acute lossof blood flow, appear to die rapidly as a result of necrosis. However,outside the central ischemic zone, cells die over a more protracted timeperiod and morphologically appear to die by apoptosis. In other orfurther embodiments, the anti-apoptotic crosslinked polypeptides of theinvention are used to treat all such disorders associated withundesirable cell death.

Some examples of immunologic disorders that are treated with thecrosslinked polypeptides described herein include but are not limited toorgan transplant rejection, arthritis, lupus, IBD, Crohn's disease,asthma, multiple sclerosis, diabetes, etc.

Some examples of neurologic disorders that are treated with thecrosslinked polypeptides described herein include but are not limited toAlzheimer's Disease, Down's Syndrome, Dutch Type Hereditary CerebralHemorrhage Amyloidosis, Reactive Amyloidosis, Familial AmyloidNephropathy with Urticaria and Deafness, Muckle-Wells Syndrome,Idiopathic Myeloma; Macroglobulinemia-Associated Myeloma, FamilialAmyloid Polyneuropathy, Familial Amyloid Cardiomyopathy, IsolatedCardiac Amyloid, Systemic Senile Amyloidosis, Adult Onset Diabetes,Insulinoma, Isolated Atrial Amyloid, Medullary Carcinoma of the Thyroid,Familial Amyloidosis, Hereditary Cerebral Hemorrhage With Amyloidosis,Familial Amyloidotic Polyneuropathy, Scrapie, Creutzfeldt-Jacob Disease,Gerstmann Straussler-Scheinker Syndrome, Bovine Spongiform Encephalitis,a prion-mediated disease, and Huntington's Disease.

Some examples of endocrinologic disorders that are treated with thecrosslinked polypeptides described herein include but are not limited todiabetes, hypothyroidism, hypopituitarism, hypoparathyroidism,hypogonadism, etc.

Examples of cardiovascular disorders (e.g., inflammatory disorders) thatare treated or prevented with the crosslinked polypeptides of theinvention include, but are not limited to, atherosclerosis, myocardialinfarction, stroke, thrombosis, aneurism, heart failure, ischemic heartdisease, angina pectoris, sudden cardiac death, hypertensive heartdisease; non-coronary vessel disease, such as arteriolosclerosis, smallvessel disease, nephropathy, hypertriglyceridemia, hypercholesterolemia,hyperlipidemia, xanthomatosis, asthma, hypertension, emphysema andchronic pulmonary disease; or a cardiovascular condition associated withinterventional procedures (“procedural vascular trauma”), such asrestenosis following angioplasty, placement of a shunt, stent, syntheticor natural excision grafts, indwelling catheter, valve or otherimplantable devices. Preferred cardiovascular disorders includeatherosclerosis, myocardial infarction, aneurism, and stroke.

EXAMPLES

The following section provides illustrative examples of the presentinvention.

Example 1 Synthesis of Crosslinked Polypeptides of Formula (I)

α-helical crosslinked polypeptides are synthesized, purified andanalyzed as previously described (Schafineister et al. (2000), J. Am.Chem. Soc. 122:5891-5892; Walensky et al (2004) Science 305:1466-70;Walensky et al (2006) Mol Cell 24:199-210) and as indicated below. Thefollowing macrocycles derived from the human BID BH3 (SEQ ID NOS107-115, respectively, in order of appearance), human BIM BH3 (SEQ IDNOS 116-122, respectively, in order of appearance) and human MAMLpeptide sequences (SEQ ID NOS 123-125, respectively, in order ofappearance) are used in this study:

TABLE 10 Compound Parent Calculated Calculated Found m/z Number PeptideSequence m/z (M + H) m/z (M + 3H) (M + 3H) 1 BIDAc-DIIRNIARHLA$VGD$NleDRSI-NH2 2438.40 813.47 813.7 2 BIDAc-DAARNIARHLA$VAibD$NleARSI-NH2 2338.35 780.12 780.17 3 BIDAc-DIARNIARHLA$VAibD$NleARSI-NH2 2380.39 794.14 794.15 4 BIDAc-DAIRNIARHLA$VAibD$NleARSI-NH2 2380.39 794.14 794.09 5 BIDPr-RNIARHLA$VAibD$NleDRSI-NH2 2139.25 713.76 713.79 6 BIDPr-RNIARHLAib$VAibD$NleDRSI-NH2 2153.27 718.43 718.5 7 BIDPr-RNIARHLA$VAibD$FARSI-NH2 2129.25 710.42 710.3 8 BIDPr-RNIARHLA$VGD$NleAibRSI-NH2 2081.25 694.42 694.42 9 BIDPr-RNIAibRHLAib$VAibD$AARSI-NH2 2081.25 694.42 694.49 10 BIMAc-IWIAQELR$IGD$FNAYYARR-NH2 2646.43 882.82 883.15 11 BIMAc-IWIAQQLR$IGD$FNAYYARR-NH2 2645.45 882.49 882.62 12 BIMAc-IWIAQALR$IGD$FNAYYARR-NH2 2588.43 863.48 863.85 13 BIMAc-RWIAQQLR$IGD$FNAYYARR-NH2 2688.46 896.83 896.84 14 BIMAc-RWIAQALR$IGD$FNAFYARR-NH2 2615.45 872.49 872.64 15 BIMAc-RWIAQALR$IGN$FNAYYARR-NH2 2630.45 877.48 877.36 16 BIMAc-IWIAQALR$IGN$FNAYYARR-NH2 2587.43 863.14 863.00 17 hMAMLAc-ERLRRRI$LCR$HHST-NH2 2124.21 709.08 708.72 18 hMAMLAc-ERLRRRI$LAR$HHST-NH2 2092.24 698.42 698.09 19 hMAMLAc- ALRRRI$LCA$HHST-NH2 1825.04 609.35 609.06

In the sequences above, compound 1, 10 and 17 are reference compoundshaving high efficacy in serum-free media, which is substantially reducedin the presence of serum. Variants of this compound (2-9, 11-16, 18-19)are then made and tested using the methods of the invention. Nlerepresents norleucine, Aib represents 2-aminoisobutyric acid, Chgrepresents cyclohexylglycine, Ac represents N-terminal acetyl, Prrepresents N-terminal proprionyl and NH₂ represents C-terminal amide.Amino acids represented as $ connect an all-carbon crosslinkercomprising eight carbon atoms between the alpha carbons of each aminoacid with a double bond between the fourth and fifth carbon atoms andwherein each α-carbon atom to which the crosslinker is attached isadditionally substituted with a methyl group. Predicted and measured m/zspectra are provided.

Alpha,alpha-disubstituted non-natural amino acids containing olefinicside chains are synthesized according to Williams et al. (1991) J. Am.Chem. Soc. 113:9276; and Schafmeister et al. (2000) J. Am. Chem Soc.122:5891. Crosslinked polypeptides are designed by replacing twonaturally occurring amino acids (see Table 10 and FIG. 5) with thecorresponding synthetic amino acids. Substitutions are made at i and i+4positions or at i and i+7 positions. Crosslinked polypeptides aregenerated by solid phase peptide synthesis followed by olefinmetathesis-based crosslinking of the synthetic amino acids via theirolefin-containing side chains.

The non-natural amino acids (R and S enantiomers of the 5-carbonolefinic amino acid and the S enantiomer of the 8-carbon olefinic aminoacid) are characterized by nuclear magnetic resonance (NMR) spectroscopy(Varian Mercury 400) and mass spectrometry (Micromass LCT). Peptidesynthesis is performed either manually or on an automated peptidesynthesizer (Applied Biosystems, model 433A), using solid phaseconditions, rink amide AM resin (Novabiochem), and Fmoc main-chainprotecting group chemistry. For the coupling of natural Fmoc-protectedamino acids (Novabiochem), 10 equivalents of amino acid and a 1:1:2molar ratio of coupling reagents HBTU/HOBt (Novabiochem)/DIEA areemployed. Non-natural amino acids (4 equiv) are coupled with a 1:1:2molar ratio of HATU (Applied Biosystems)/HOBt/DIEA. Olefin metathesis isperformed in the solid phase using 10 mM Grubbs catalyst (Blackewell etal. 1994 supra) (Materia) dissolved in degassed dichloromethane andreacted for 2 hours at room temperature. Isolation of metathesizedcompounds is achieved by trifluoroacetic acid-mediated deprotection andcleavage, ether precipitation to yield the crude product, and highperformance liquid chromatography (HPLC) (Varian ProStar) on a reversephase C18 column (Varian) to yield the pure compounds. Chemicalcomposition of the pure products is confirmed by LC/MS mass spectrometry(Micromass LCT interfaced with Agilent 1100 HPLC system) and amino acidanalysis (Applied Biosystems, model 420A).

Example 2 Cell Viability Assays of Tumor Cell Lines Treated withCrosslinked Polypeptides of the Invention

Jurkat cell line (Clone E6-1, ATCC catalog #TIB-152) is grown inspecific serum-supplemented media (RPMI-1640, Invitrogen catalog #22400)as recommended by ATCC. A day prior to the initiation of the study,cells are split at optimal cell density (2×10⁵-5×10⁵ cells/ml) to assureactively dividing cells. The next day, cells are washed twice inserum-free Opti-MEM media (Invitrogen, Catalog #51985) and cells arethen plated at optimal cell density (10,000 cells/well) in 50 μlOpti-MEM media or Opti-MEM supplemented with 2% or 10% human serum(Bioreclamation, catalog #HMSRM) in 96-well white tissue culture plate(Nunc, catalog #136102).

For serum free experiment, crosslinked polypeptides are diluted from 2mM stocks (100% DMSO) in sterile water to prepare 400 μM workingsolutions. The crosslinked polypeptides and controls are diluted 10-foldfirst and then serially two-fold diluted in Opti-MEM in dosing plates toprovide concentrations of between 1.2 and 40 μM. 50 μL of each dilutionis then added to the appropriate wells of the test plate to achievefinal concentrations of the polypeptides equal to between 0.6 to 20 μM.For studies using Opti-MEM supplemented with human serum(Bioreclamation, catalog #HMSRM), crosslinked polypeptides are dilutedfrom 10 mM stocks (100% DMSO) in sterile water to prepare 2 mM workingsolutions. The crosslinked polypeptides and controls are diluted 10-foldfirst and then serially two-fold diluted in Opti-MEM in the presence of2% or 10% of human serum to provide concentrations of the polypeptidesequal to between 6.25 to 200 μM in dosing plates. 50 μL of each dilutionis then added to the appropriate wells of the test plate to achievefinal concentrations of the polypeptides equal to between 3.125 to 100μM. Controls included wells without polypeptides containing the sameconcentration of DMSO as the wells containing the macrocycles, wellscontaining 0.1% Triton X-100 and wells containing no cells. Plates areincubated for 24 hours at 37° C. in humidified 5% CO₂ atmosphere.

At the end of the incubation period, CellTiter-Glo assay is performedaccording to manufacturer's instructions (Promega, catalog #G7573) andluminescence is measured using Synergy HT Plate reader (BioTek).Luminescence correlates with viability. A reduction in viabilityreflects the ability of the test compounds to induce programmed celldeath via BAX and BAK. A representative dose-response curve atincreasing concentrations of human serum is shown in FIG. 1.

Example 3 Determination of Apparent Affinity to Human Serum Proteins(K_(d)*)

The measurement of apparent K_(d) values for serum protein by EC50 shiftanalysis provides a simple and rapid means of quantifying the propensityof experimental compounds to bind HSA and other serum proteins. A linearrelationship exists between the apparent EC₅₀ in the presence of serumprotein (EC′₅₀) and the amount of serum protein added to an in vitroassay. This relationship is defined by the binding affinity of thecompound for serum proteins, expressed as K_(d)*. This term is anexperimentally determined, apparent dissociation constant that mayresult from the cumulative effects of multiple, experimentallyindistinguishable, binding events. The form of this relationship ispresented here in Eq. 0.3, and its derivation can be found in Copelandet al, Biorg. Med Chem Lett. 2004, 14:2309-2312.

$\begin{matrix}{{EC}_{50}^{\prime} = {{EC}_{50} + {P\left( \frac{n}{1 + \frac{K_{d}^{*}}{{EC}_{50}}} \right)}}} & (0.3)\end{matrix}$

A significant proportion of serum protein binding can be ascribed todrug interactions with HSA, due to the very high concentration of thisprotein in serum (35-50 g/L or 530-758 μM). To calculate the K_(d) valuefor these compounds we have assumed that the shift in EC₅₀ upon proteinaddition can be ascribed fully to the HSA present in the added serum,where P is 700 μM for 100% serum, P is 70 μM for 10% serum, etc. Wefurther made the simplifying assumption that all of the compounds bindHSA with a 1:1 stoichiometry, so that the term n in Eq. (0.3) is fixedat unity. With these parameters in place we calculated the K_(d)* valuefor each stapled peptide from the changes in EC₅₀ values with increasingserum (and serum protein) concentrations by nonlinear regressionanalysis of Eq. 0.3 using Mathematica 4.1 (Wolfram Research, Inc.,www.wolfram.com). EC′₅₀ values in whole blood are estimated by setting Pin Eq. 0.3 to 700 μM [HSA].

The free fraction in blood is estimated per the following equation, asderived by Trainor, Expert Opin. Drug Disc., 2007, 2(1):51-64, where[HSA]_(total) is set at 700 μM.

$\begin{matrix}{{FreeFraction} = \frac{K_{d}^{*}}{K_{d}^{*} + \lbrack{HSA}\rbrack_{total}}} & (0.4)\end{matrix}$

FIG. 2 shows representative plots of EC50 vs human serum concentrationfor compound 1 and related analogs. FIG. 3 shows representative plots ofEC50 vs human serum concentration for compound 10 and related analogs.

Table 11 shows that by selection and optimization in accordance with theinvention, compounds can be made with substantially less serum shiftthan the initial lead (for example, compound 1 or compound 10) whilestill retaining good activity in the assay of Example 2.

TABLE 11 Compound No serum 2% serum 10% serum Free Fraction est. EC50est. Number EC50, μM EC50, μM EC50, μM Serum Kd* in blood, μM in blood,μM 1 1.2 73.9 >100 <0.1 <0.1% 3636.2 2 1.6 20.5 97.1 <0.1 <0.1% 957.2 31.2 14.9 88.9 <0.1 <0.1% 890.1 4 1.0 11.3 73.1 <0.1 <0.1% 734.8 5 1.616.7 63.0 0.2 0.04% 606.9 6 1.0 10.1 49.7 0.4 0.07% 490.0 7 2.2 12.448.2 1.1 0.19% 459.1 8 2.4 10.5 37.7 2.3 0.39% 352.2 9 1.1 5.6 20.2 2.90.48% 190.0 10 1.3 36.9 >100 <0.1 <0.1% 1781.3 11 1.2 7.9 37.6 1.1 0.18%367.0 12 1.3 8.6 26.5 2.3 0.38% 246.3 13 1.5 5.4 21.5 3.8 0.63% 201.8 140.4 2.8 10.8 2.3 0.38% 103.4 15 0.9 2.6 11.5 5.1 0.84% 108.2 16 0.5 2.39.3 3.5 0.58% 88.4 17 12.0 55.1 >100 <0.1 <0.1% 2167.0 18 >20 >100 >100<0.1 <0.1% >4000 19 2.4 14.7 57.5 0.6 0.10% 549.4

Example 4 Structure-Activity Relationship of the Apparent Affinity toHuman Serum Proteins (K_(d)*)

FIG. 4 shows helical wheel representations of crosslinked peptide pairsof the invention in which one or more amino acids is altered to providea crosslinked peptide analog with improved efficacy towardsintracellular target(s) in whole cell assays. Across a number ofsequences it is observed that a dipeptide motif consisting of an acidic(negatively charged) side chain adjacent to a large hydrophobic sidechain yields higher affinity binding to human serum proteins such asalbumin relative to an analog in which the acidic side chain has beenreplaced with a neutral side chain. In some cases replacement of boththe acidic and large hydrophobic side chains with neutral and lesshydrophobic side chains, respectively, provides lower affinity to humanserum proteins. This structure activity relationship is consistent withthe understanding that human serum proteins, and in particular humanserum albumin, bind fatty acids under physiological conditions, andthese fatty acids are recognized by a combined acidic/hydrophobicbinding motif. It is also known that the membranes of human and animalcells consist of phospholipids and that the phosphate head groups of thelipid bilayer present a negatively charged surface at the outer membranethat will electrostatically repulse acidic (negatively charged) sidechains of a peptide, and thus the replacement of an acidic side chainwith a neutral side chain should increase the association of acrosslinked peptide with the cell membrane. This association with theouter membrane is the proposed required first step in the endocytosis ofthe crosslinked peptides of the invention.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

What is claimed is:
 1. A method of screening for an enhancedalpha-helical polypeptide with enhanced cellular efficacy in human wholeblood, the enhanced alpha-helical polypeptide comprising a cross-linkerconnecting a first amino acid and a second amino acid of thepolypeptide, the method comprising: (a) providing a sequence of a parentpolypeptide comprising a cross-linker connecting a first amino acid anda second amino acid of the parent polypeptide sequence, (b) producing amodified alpha-helical polypeptide having the same sequence as theparent polypeptide sequence except that at least one amino acid sidechain that is not essential for target binding is different incomparison to the alpha-helical parent polypeptide sequence, wherein themodified alpha-helical polypeptide comprises a cross-linker connecting afirst amino acid and a second amino acid of the modified alpha-helicalpolypeptide; (c) measuring an in vitro activity (EC₅₀) of the modifiedalpha-helical polypeptide in a whole cell assay wherein activity ismediated by binding to a target, in the presence and absence of humanserum; (d) determining an apparent affinity (K_(d)*) of the modifiedalpha-helical polypeptide for human serum proteins; and (e) selectingthe modified alpha-helical polypeptide as an enhanced alpha-helicalpolypeptide if the modified alpha-helical polypeptide has a K_(d)*between about 1 and 700 micromolar.
 2. The method of claim 1, whereinthe enhanced alpha-helical polypeptide has a K_(d)* between about 1 and70 micromolar.
 3. The method of claim 1, wherein the enhancedalpha-helical polypeptide has a K_(d)* of about 1-10 micromolar.
 4. Themethod of claim 1, wherein the enhanced alpha-helical polypeptidepossesses an estimated free fraction in human blood of about 0.1-50%,wherein the estimated free fraction is defined by the equation${FreeFraction} = \frac{K_{d}^{*}}{K_{d}^{*} + \lbrack{HSA}\rbrack_{total}}$and [HSA]_(total) is 700 micromolar.
 5. The method of claim 1, whereinthe enhanced alpha-helical polypeptide possesses an estimated freefraction in human blood of about 0.5-10%.
 6. The method of claim 1,wherein at least one of the first and second amino acids is anα,α-disubstituted amino acid.
 7. The method of claim 1, wherein both thefirst and second amino acids are α,α-disubstituted.
 8. The method ofclaim 1, wherein the enhanced alpha-helical polypeptide comprises 1 or 2turns of an alpha-helix.
 9. The method of claim 1, wherein the firstamino acid and the second amino acid are separated by three amino acids.10. The method of claim 1, wherein the cross-linker comprises between 6and 14 consecutive bonds.
 11. The method of claim 1, wherein thecross-linker comprises between 8 and 12 consecutive bonds.
 12. Themethod of claim 1, wherein the enhanced alpha-helical polypeptidecomprises a ring of about 18 atoms to 26 atoms.
 13. The method of claim1, wherein the first amino acid and the second amino acid are separatedby six amino acids.
 14. The method of claim 1, wherein the cross-linkercomprises between 8 and 16 consecutive bonds.
 15. The method of claim 1,wherein the cross-linker comprises between 10 and 13 consecutive bonds.16. The method of claim 1, wherein the enhanced alpha-helicalpolypeptide comprises a ring of about 29 atoms to 37 atoms.
 17. Themethod of claim 1, wherein the length of the cross-linker is about 5 Åto about 9 Å per turn of an alpha-helix.
 18. The method of claim 1,wherein the enhanced alpha-helical polypeptide carries a net positivecharge at pH 7.4.
 19. The method of claim 1, wherein the enhancedalpha-helical polypeptide comprises one or more of a halogen, an alkylgroup, a fluorescent moiety, an affinity label, a targeting moiety, or aradioisotope.
 20. The method of claim 1, wherein the enhancedalpha-helical polypeptide provides a therapeutic effect.
 21. The methodof claim 1, wherein the enhanced alpha-helical polypeptide possesses anapparent affinity to human serum proteins of about 1 micromolar orweaker.
 22. The method of claim 1, wherein the enhanced alpha-helicalpolypeptide possesses an apparent affinity to human serum proteins ofabout 3 micromolar or weaker.
 23. The method of claim 1, wherein theenhanced alpha-helical polypeptide possesses an apparent affinity tohuman serum proteins of about 10 micromolar or weaker.
 24. The method ofclaim 1, wherein K_(d)* is defined by the equation${EC}_{50}^{\prime} = {{EC}_{50} + {P\left( \frac{n}{1 + \frac{K_{d}^{*}}{{EC}_{50}}} \right)}}$wherein n is 1, EC₅₀ is an in vitro efficacy measured in a whole cellassay in the absence of any human serum, and EC′₅₀ is an in vitroefficacy measured in a whole cell assay in N % human serum wherein Pequals (N/100)×(700) micromolar.
 25. A polypeptide comprising across-linker connecting a first amino acid and a second amino acid ofthe polypeptide, wherein the polypeptide penetrates cell membranes by anenergy-dependent process and binds to a target, and wherein thepolypeptide is selected according to the method of claim 1.